Presence Of Prostaglandin Research Articles

GRK2 Regulates ADP Signaling in Platelets Via P2Y 1 and P2Y 12

AbstractVenous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA 2, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y 1 and P2Y 12. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation.Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 -/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA 2, or convulxin. Underlying these changes in GRK2 -/- platelets is an increase in Ca 2+ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I 2. Furthermore, platelet aggregation can be restored in GRK2 -/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y 12 signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 -/- mice treated with the P2Y 12 antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 -/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation.Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y 1 and P2Y 12-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases. DisclosuresNo relevant conflicts of interest to declare.

Modulation of Peripheral Inflammatory Pain Thresholds by M1 and Nicotinic Receptor Antagonists

The study used the paw withdrawal test to investigate the role of the cholinergic system on the modulation of inflammatory pain induced by carrageenan (Cg) at the peripheral level, through activation of muscarinic and nicotinic receptors. Intraplantar administration of the specific M₁ receptor antagonist telenzepine (TEL; 6, 12 and 24 µg/paw) caused a dose-dependent reduction in the nociceptive threshold induced by Cg (125 µg/paw). This effect was not observed with increasing doses (4, 10 and 40 µg) of other specific receptor antagonists: M₂ (dimethindene), M₃ (4-DAMP) and M₄ (tropicamide). The nicotinic antagonist mecamylamine (MEC; 25, 50 and 100 µg/paw) also caused a dose-dependent reduction in the nociceptive threshold induced by Cg (125 µg). To exclude a non-local effect, Cg (125 µg) was injected into both hind paws, while TEL (12 µg) and MEC (50 µg) were administered only in the right paw. At these doses, the muscarinic antagonists increased inflammatory pain only in the treated right paw, suggesting a peripheral effect. In the presence of prostaglandin E₂ (1 µg/paw), TEL (12 µg) and MEC (50 µg) did not reduce the nociceptive threshold, suggesting that this hyperalgesic agent does not induce the release of endogenous acetylcholine. These data suggest that muscarinic M₁ and nicotinic receptors participate in the modulation of endogenous cholinergic inflammatory pain at the peripheral level.

Comparison of VerifyNow-P2Y12 test and Flow Cytometry for monitoring individual platelet response to clopidogrel. What is the cut-off value for identifying patients who are low responders to clopidogrel therapy?

BackgroundDual anti-platelet therapy with aspirin and a thienopyridine (DAT) is used to prevent stent thrombosis after percutaneous coronary intervention (PCI). Low response to clopidogrel therapy (LR) occurs, but laboratory tests have a controversial role in the identification of this condition.MethodsWe studied LR in patients with stable angina undergoing elective PCI, all on DAT for at least 7 days, by comparing: 1) Flow cytometry (FC) to measure platelet membrane expression of P-selectin (CD62P) and PAC-1 binding following double stimulation with ADP and collagen type I either in the presence of prostaglandin (PG) E1; 2) VerifyNow-P2Y12 test, in which results are reported as absolute P2Y12-Reaction-Units (PRU) or % of inhibition (% inhibition).ResultsThirty controls and 52 patients were analyzed. The median percentage of platelets exhibiting CD62P expression and PAC-1 binding by FC evaluation after stimulation in the presence of PG E1 was 25.4% (IQR: 21.4–33.1%) and 3.5% (1.7–9.4%), respectively. Only 6 patients receiving DAT (11.5%) had both values above the 1st quartile of controls, and were defined as LR. Evaluation of the same patients with the VerifyNow-P2Y12 test revealed that the area under the receiver-operating-characteristic (ROC) curve was 0.94 (95% CI: 0.84–0.98, p < 0.0001) for % inhibition and 0.85 (0.72–0.93, p < 0.005) for PRU. Cut-off values of ≤ 15% inhibition or > 213 PRU gave the maximum accuracy for the detection of patients defined as having LR by FC.ConclusionIn conclusion our findings show that a cut-off value of ≤ 15% inhibition or > 213 PRU in the VerifyNow-P2Y12 test may provide the best accuracy for the identification of patients with LR.

Effects of Oxidative Stress in Trabecular Meshwork Cells Are Reduced by Prostaglandin Analogues

The trabecular meshwork (TM) of glaucomatous eyes is characterized by cell loss, increased accumulation of extracellular matrix (ECM), and cellular senescence. One factor increasingly discussed in the pathogenesis of primary open-angle glaucoma (POAG) is oxidative stress. The goal of this study was to determine whether oxidative stress is able to trigger these typical glaucomatous changes in vitro and whether these oxidative stress-induced TM changes can be reduced by the application of prostaglandin analogues. Cultured human TM cells were exposed to 200 to 800 microM hydrogen peroxide (H(2)O(2)) for 1 hour. Cell loss was detected by cell-viability assay. Levels of fibronectin and MMP-2 mRNA were determined by real-time PCR analysis. Senescence-associated beta-galactosidase (SA-beta-Gal) activity was investigated by histochemical staining. The effects of prostaglandin analogues and benzalkonium chloride (BAC) on these glaucoma typical TM changes were investigated by preincubation of nonstressed or H(2)O(2)-treated cells with 1:100 diluted commercial solutions of bimatoprost, travoprost, and latanoprost or their corresponding BAC concentrations. H(2)O(2) induced cell death and fibronectin mRNA expression, but decreased the amount of MMP-2 mRNA. H(2)O(2) increased SA-beta-Gal activity. Additional pretreatment with BAC further increased the typical glaucomatous TM changes in vitro. These effects were reduced by preincubation with prostaglandin analogues in H(2)O(2)-treated and, to a lesser extent, in nonstressed cells. No reduction occurred in the presence of prostaglandin F receptor antagonists in H(2)O(2)-treated cells. Oxidative stress is able to induce characteristic glaucomatous TM changes in vitro, and these oxidative stress-induced TM changes can be minimized by the use of prostaglandin analogues. Thus, prevention of oxidative stress exposure to the TM may help to reduce the progression of POAG.

Hierarchy of eosinophil chemoattractants: role of p38 mitogen‐activated protein kinase

Several chemoattractants can regulate the recruitment of eosinophils to sites of inflammation, but the hierarchy among them is unknown. We observed here that eosinophil chemotaxis towards eotaxin or 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) was amplified up to sixfold in the presence of prostaglandin (PG) D2. This effect was only seen in eosinophils, and not in neutrophils or basophils. Pretreatment with the chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) antagonist ramatroban prevented the PGD2 enhancement of eosinophil migrations. In contrast, eotaxin or 5-oxo-ETE inhibited the migration of eosinophils towards PGD2. 5-oxo-ETE enhanced the chemotaxis to eotaxin, while eotaxin had no effect on 5-oxo-ETE-induced migration. 5-oxo-ETE induced the phosphorylation of p38 mitogen-activated protein kinase, and inhibition of p38 mitogen-activated protein kinase by SB-202190 converted the effect of 5-oxo-ETE on the chemotaxis to PGD2 from inhibition to enhancement. The presence of blood or plasma markedly decreased the sensitivity of eosinophils to eotaxin or 5-oxo-ETE, while responses to PGD2 were unaltered. In conclusion, PGD2 might be an initial chemoattractant, since it maintains its potency in the circulation and augments the responsiveness of eosinophils to other chemoattractants. In contrast, eotaxin seems to be an end-point chemoattractant, since it has reduced efficacy in blood and is capable of down-modulating eosinophil responsiveness to other chemoattractants.

Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE(2)) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE(2) and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE(2) induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE(2) receptors EP2 and EP4 eliminated the PGE(2)-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE(2)-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE(2) response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE(2) stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE(2) plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.

CCL19/CCL21-triggered signal transduction and migration of dendritic cells requires prostaglandin E2

AbstractThe control of dendritic cell (DC) migration is pivotal for the initiation of cellular immune responses. When activated with inflammatory stimuli, the chemokine receptor CCR7 is up-regulated on DCs. Activated DCs home to lymphoid organs, where the CCR7 ligands CCL19 and CCL21 are expressed. We previously found that human monocyte-derived DCs (MoDCs) exclusively migrated to CCL19 and CCL21 when matured in the presence of prostaglandin (PG) E2. Because PGE2 did not alter CCR7 cell surface expression, we examined whether PGE2 may exert its effect by coupling CCR7 to signal transduction modules. Indeed, stimulation with CCR7 ligands led to enhanced phosphatidylinositol-3-kinase–mediated phosphorylation of protein kinase B when MoDCs were matured in the presence of PGE2. Moreover, CCL19/CCL21-induced intracellular calcium mobilization in MoDCs occurred only when PGE2 was present during maturation. MoDC migration to CCL19 and CCL21 was dependent on phospholipase C and intracellular calcium flux but not on phosphatidylinositol-3 kinase. Hence, our data provide insight into CCL19/CCL21-triggered signal transduction pathways and identify a novel function for PGE2 in controlling the migration of mature MoDCs by facilitating CCR7 signal transduction.

Adipocyte differentiation of multipotent cells established from human adipose tissue

In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of β-adrenoreceptors (β2-AR agonist > β1-AR agonist ≫ β3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F 2α, tumour necrosis factor-α, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism.

Hypoxia induces the release of a pulmonary-selective, Ca(2+)-sensitising, vasoconstrictor from the perfused rat lung.

Sustained hypoxic pulmonary vasoconstriction is dependent upon the presence of an intact endothelium, strongly suggesting that an endothelium-derived constrictor factor is involved in this response. In the present study we have attempted to determine whether hypoxia induces the release of a vasoconstrictor(s) from the lung, and whether this vasoconstrictor shares mechanistic features with the hypoxic constrictor response. The salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat intrapulmonary artery functional bioassay were utilised in this study. Hypoxic, but not normoxic, perfusion of the isolated lung of the rat induced the release of a vasoconstrictor(s) which appeared to be selective for pulmonary over mesenteric arteries of the rat. The vasoconstriction observed was unaffected by inhibition of voltage-gated Ca(2+) channels, and was not associated with a rise in intracellular [Ca(2+)], suggesting Ca(2+)-sensitisation of the contractile apparatus. The vasoconstriction was also unaffected by the protein kinase C (PKC) inhibitor Ro-31-8220, or the endothelin-1 antagonists BQ123/BQ788 but was markedly potentiated in the presence of prostaglandin F(2alpha). We conclude that hypoxic perfusion of the rat lung results in the release of a vasoconstrictor(s) which shares some of the facets of the sustained hypoxic constriction of isolated intrapulmonary arteries of the rat, since it involves PKC-independent Ca(2+) sensitisation, is independent of voltage-gated Ca(2+) entry, and is potentiated by the presence of preconstriction.

Chronic nitric oxide inhibition model six years on.

The discovery in 1987 that endothelium-derived nitric oxide (NO) mediates the vasodilatory effect of certain endothelium-dependent agonists1 2 inaugurated the current huge field of NO biology. It is now recognized that NO plays essential roles in many diverse physiological processes and in some pathophysiologic events. Development of these concepts has been based largely on evidence obtained by limiting NO biosynthesis. This review is centered on the cardiovascular and particularly the renal functional and structural consequences of chronic pharmacologic NO inhibition by l-arginine analogues. We devoted special attention to the mechanisms of hypertension and organ injury that occur under these circumstances, while appreciating the inherent limitations surrounding interpretation of this data. NO is made by the enzymatic action of several widely distributed NO synthases (NOS). In the presence of the substrates l-arginine and oxygen, as well as a number of essential cofactors, NO is produced in response to appropriate stimuli. The constitutively expressed NOS play a major role in the physiological control of vascular tone and kidney function.3 4 Vascular endothelial NOS (eNOS) and brain-type NOS (bNOS) are widely distributed throughout the kidney5 as well as the cardiovascular system and in strategic locations in the peripheral and central nervous system (CNS).6 7 Both eNOS and particularly bNOS are abundant in the kidney, glomeruli, and vasculature as well as in most segments of the tubule,3 5 and NOS activity in medulla is considerably greater than in cortex.8 NO generated within the kidney controls the glomerular filtration rate (GFR), total renal and medullary blood flow, pressure natriuresis, epithelial sodium transport, and production of various vasoactive factors including renin.3 4 5 eNOS is distributed throughout most parts of the arterial and venous circulation, although there is considerable heterogeneity in the extent to which NO controls …

Cyclic AMP-levels of retinoic acid primed HL-60 cells in serum-free medium influenced by a 50 Hz magnetic field alone and as co-factor to prostaglandin E 2

In the present study, the human cell line HL-60 was induced to differentiation to mature granulocytes by the treatment with retinoic acid and prostaglandin E 2 in physiological concentration. During the process of priming with retinoic acid and differentiation with prostaglandin E 2 changes in the concentration of the intracellular second messenger, cyclic adenosin monophosphate, plays a certain role among changes in the other signal transduction pathways. Analysis of the time sequence of cyclic adenosin monophosphate levels showed that in serum-free medium the concentration temporarily increased, when HL-60 cells were incubated for 5 min with 10 nM retinoic acid. 24 h later the cells are on control level again. Priming with retinoic acid had no lasting influence on the rate of cell proliferation. When retinoic acid primed cells were incubated later for 5 min with 10 nM prostaglandin E 2, their cyclic adenosin monophosphate levels increased more pronounced. In the following 72–96 h, the cell proliferation was slowed down due to this treatment with prostaglandin E 2. In comparison, a single exposure of retinoic acid primed cells for 5 min to a 50 Hz magnetic field with 2 mT flux density led to a small, but statistical significant increase of the cyclic adenosin monophosphate level, similar to that observed after the treatment with retinoic acid, 24 h before. In combination with prostaglandin E 2, the magnetic field superimposed the cyclic adenosin monophosphate induced rise due to prostaglandin E 2, acting in an additive way. Finally, when HL-60 cells were once treated with the 2 mT magnetic field for 60 min, their growth rate was reduced without a loss of cell viability, but not until 144 h after the field treatment. This was found in both absence and presence of prostaglandin E 2. In conclusion, the magnetic field produced a small, but similar effect compared to that of prostaglandin E 2 and acted as an additive, when used as co-factor.

Characterization of histamine receptors in the ureter of the dog

We investigated the effects of histamine on the motility of isolated segments from canine ureters and characterized pharmacologically the histamine receptors involved. We also evaluated the effects of various autacoids (5-HT, carbachol, noradrenaline, thromboxane, prostaglandin F 2α) on the motility of canine ureters. Histamine as well as the H 1 receptor agonist 2-(2-pyridyl)ethylamine elicited a concentration-dependent contraction. This contractile response was antagonized by dimethindene, causing a rightward shift (pA 2 8.30) and a reduction of the slope and the maximal effect (pD′ 2 6.01) of the concentration-response curve. The histamine H 2 receptor antagonist cimetidine in a concentration of 10 −5 mol/l was ineffective concerning the concentration-response curve for histamine. After precontraction of the ureter segments (5-HT, carbachol, prostaglandin F 2α), a concentration-dependent relaxant effect was evaluated in the presence of histamine or the histamine H 2 receptor agonist impromidine. The histamine H 2 receptor antagonist cimetidine attenuated the relaxant response, causing a rightward shift of the concentration-response curve. All autacoids except thromboxane were capable of increasing contractility in canine ureters. Comparing the absolute contractile force in the presence of prostaglandin F 2α. 5-HT, carbachol, noradrenaline and potassium, we found that histamine exhibits the most marked effect on this parameter in the canine ureter. It is concluded that there are two types of histamine receptors modulating contractile activity in the canine ureter: histamine H 1 receptors, which mediate contraction, and histamine H 2 receptors, which mediate relaxation (in the precontracted tissue).

Cyclooxygenases-1 and −2 of Endothelial Cells Utilize Exogenous or Endogenous Arachidonic Acid for Transcellular Production of Thromboxane

The presence of prostaglandin (PG) H2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF1alpha, PGE2, PGF2alpha) versus transcellular (TXB2) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1alpha, newly synthesized Cox-2 produced less TXB2 than 6-keto-PGF1alpha compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.

Effects of prostaglandins and cyclic AMP on cytokine production in rat leukocytes

Prostaglandins E 1, prostaglandin E 2, 3-oxa-methano-prostaglandin I 1 (SM-10906), a stable prostaglandin I 2 analog, and dibutyryl cyclic AMP suppressed the production of tumor necrosis factor and interleukin-1 in lipopolysaccharide-stimulated rat pleural resident monocytic cells, whereas they enhanced the production of interleukin-6 and cytokine-induced neutrophil chemoattractant (CINC), a rat interleukin-8-like chemokine, in these cells. SM-10906 also inhibited the in vivo production of tumor necrosis factor and interleukin-1 in pleural exudates, when injected into the rat pleural cavity concomitantly with carrageenin. The cyclic AMP (cAMP) level in the lipopolysaccharide-stimulated resident cells was increased when the cells were incubated in the presence of prostaglandin E 1, prostaglandin E 2 or SM-10906. Prostaglandin I 2 showed only slight effects. The addition of pentoxifylline, a phosphodiesterase inhibitor, to the incubation mixture increased the cAMP level and also enhanced the effect of prostaglandins, indicating that these regulating actions of prostaglandins may be exerted partly through a mechanism involving an increased intracellular cAMP level.

Oesophagostomum dentatum: Population Dynamics and Synthesis of Prostanoids by Histotropic Stages Cultured in Vitro

The dynamics of production and excretion of prostanoids by histotropic larvae of Oesophagostromum dentatum and the role of prostanoids for the development of O. dentatum were studied under in vitro conditions. In control cultures fourth stage larvae (L4) were first seen on Day 7 of in vitro cultivation. Their numbers steadily increased until Day 21 of in vitro cultivation. Thereafter, the numbers of IA no further increased. The continuous presence of inhibitors of prostanoid synthesis such as acetylsalicylic acid (ASA, 5.6 mmole/liter) or indomethacin (INDO, 1.4 mmole/liter) in the culture medium blocked the development to L4 almost completely. When ASA was given first on Day 13 of in vitro cultivation L4 counts still increased during the following 4 days. However, the numbers of larvae which reached the L4 stage in these cultures was reduced to 65% of that in control cultures. Resumption of development to L4 was seen after the withdrawal of INDO in cultures treated with this drug until Day 13 of in vitro, cultivation. Larvae and supernatants were collected separately on Days 7, 14, 21, or 28 of in vitro cultivation and larvae were homogenized. The presence of prostaglandin (PG) E2, PGD2, PGF2α, 6-keto-PGF1α and thromboxane (TX) B2 was assayed radioimmunologically in homogenates and supernatants. In the homogenates only PGE2 and PGD2 were regularly detected. The total amount of PGE2 and PGD2 in the homogenates significantly increased with the increase in L4 numbers, whereas the homogenate protein-based values did not. In addition to PGD2 and PGE2, PGF2α and TXB2 were found in the supernatants. PGE2 accounted for about 50% of the prostanoids in the supernatants on Day 7 of in vitro cultivation but was generally absent thereafter. After 14, 21, and 28 days of in vitro cultivation TXB2 was the most prominent single prostanoid in the supernatants. PGD2 was found in the supernatants only after 7 and 28 days of in vitro cultivation. PGF2α was present in decreasing amounts in the supernatants throughout the cultivation period. In conclusion, histotropic larvae of O. dentatum produce and excrete mediators of the prostanoid type which may serve as endogenous hormone-like growth factors.

A novel function of haptoglobin: haptoglobin-haemoglobin complex induces apoptosis of hepatocarcinomatous Hep 3B cells

Haptoglobin-haemoglobin complex (Cx) showed a cytotoxic effect on the growth of Hep 3B (human hepatocellular carcinoma) cells, dose dependently. The antiproliferative effect of Cx on the multiplication of Hep 3B cells was augmented by the presence of prostaglandin (PG) D2. Antihuman Hb IgG abolished the effect of Cx, dose-dependently, which indicates that the antiproliferative effect of Cx really is exerted by Cx. Hep 3B cells treated with Cx showed the characteristic biochemical changes of apoptosis, such as DNA fragmentation which was blocked by pretreatment with cycloheximide, and the increase of transglutaminase expression. Thus, the antiproliferative effect of Cx against Hep 3B cells occurs via the typical apoptotic pathway.

Enzymatic Oxidation of Dopamine: The Role of Prostaglandin H Synthase

An enzyme responsible for the oxidation of dopamine and formation of neuromelanin in brain has not been identified. Prostaglandin H synthase is prominent in brain and possesses peroxidase activity that may cooxidize dopamine to reactive dopamine quinones. This study examined the ability of purified prostaglandin H synthase to catalyze the oxidation of dopamine in vitro. Dopamine oxidation was determined by monitoring the formation of aminochrome and by examining catechol-modified residues on protein present in the reaction mixture. Aminochrome was formed from dopamine in the presence of prostaglandin H synthase, and the reaction rate was dependent on the concentration of substrate and enzyme in the reaction mixture. Both arachidonic acid and hydrogen peroxide could serve as substrates for the prostaglandin H synthase-catalyzed oxidation of dopamine. Indomethacin blocked the reaction when arachidonic acid was used as a substrate, but not when hydrogen peroxide was used. Enzymatically oxidized dopamine covalently bound to protein, as indicated by the presence of cysteinyl-dopamine residues. Binding was significantly reduced in the absence of enzyme or in the presence of antioxidants. These results suggest that the peroxidase activity of prostaglandin H synthase is responsible for catalyzing the oxidation of dopamine to reactive dopamine quinones. It is possible that prostaglandin H synthase is responsible for the oxidation of dopamine and formation of neuromelanin in vivo, which may have implications for the development of Parkinson's disease. Furthermore, drugs such as aspirin that modulate the activity of this enzyme may provide a potential therapeutic approach for the prevention of Parkinson's disease.

Nitric oxide release from resting human platelets

In this study, we have investigated the release of nitric oxide from resting human platelets. Nitric oxide was detected and quantitated by either measuring the conversion of oxy-hemoglobin to met-hemoglobin or generation of nitrite and nitrate by the cells. Nitric oxide was released from both intact resting platelets and platelets activated by collagen. Nitric oxide release was proportional to platelet concentration, and was equivalent to approximately 4.5 ± 0.6 pmol (or 2.8 ± 0.3 pmol in the presence of prostaglandin I 2) and 11.2 ± 1.3 pmol nitric oxide released per minute per 10 8 cells at 37 °C for resting platelets and platelets activated by collagen, respectively. The generation of nitric oxide by resting platelets was linear with respect to time over a two hour period, while the release of nitric oxide from platelets following activation was transient and was linear for only the first 10 min, after which it slowed to completion at approximately 30 min. The release of nitric oxide was stimulated by L-arginine, but was inhibited by L-nitro-arginine methyl ester (L-NAME). The inhibitory effect of L-NAME could be reversed by addition of L-arginine. The release of nitric oxide from platelets was also partially inhibited by prostaglandin I 2, prostaglandin E 1, aspirin and EDTA. The amount of nitric oxide released from resting platelets compared with that released from endothelial cells suggests that platelet-derived nitric oxide may play a significant role in the maintenance of vascular tone and blood flow.

A novel function of haptoglobin: haptoglobin-haemoglobin complex induces apoptosis of hepatocarcinomatous Hep 3B cells.

Haptoglobin-haemoglobin complex (Cx) showed a cytotoxic effect on the growth of Hep 3B (human hepatocellular carcinoma) cells, dose dependently. The antiproliferative effect of Cx on the multiplication of Hep 3B cells was augmented by the presence of prostaglandin (PG) D2. Antihuman Hb IgG abolished the effect of Cx, dose-dependently, which indicates that the antiproliferative effect of Cx really is exerted by Cx. Hep 3B cells treated with Cx showed the characteristic biochemical changes of apoptosis, such as DNA fragmentation which was blocked by pretreatment with cycloheximide, and the increase of transglutaminase expression. Thus, the antiproliferative effect of Cx against Hep 3B cells occurs via the typical apoptotic pathway.

Potentiation of norepinephrine-induced contraction by primary prostaglandin receptor activation in rat aorta

This study investigates the role of primary prostaglandin receptor activation in the modulation of agonist-induced vascular smooth muscle contraction. Prostaglandin F 2α induced a concentration-dependent contraction of the rat aorta that was nearly abolished by the thromboxane A 2 receptor antagonist, SQ29548. Prostaglandin F 2α in the presence of SQ29548 induced leftward shifts of the norepinephrine and KCl concentration-response curves. Nifedipine abolished the leftward shift of the norepinephrine concentration-response curve observed in the presence of prostaglandin F 2α and SQ29548. These results suggest that a function of primary prostaglandin receptor activation may be to potentiate agonist-induced contraction. The potentiation is dependent upon the opening of dihydropyridine-sensitive Ca 2+ channels.