Vascular smooth muscle activity of 7-oxa-13-prostynoic acid

The effects of 7-oxa-13-prostynoic acid (7-OPA), alone and as an antagonist of PGE2 and PGF2alpha, were investigaed in isolated rabbit aortic and canine renal arterial (diameter approximentaly 0.5 mm) strips. 7-OPA caused contractions in both preparations; threshold concentrations were 3- to 10-fold higher than the PGs and maximum contraction was 50-60%. In concentrations of 10(-5) M, 7-OPA inhibited contractions by PGF2alpha and PGE2. The same concentration of 7-OPA did not inhibit norepinephrine-induced contractions. These data indicate that 7-OPA is a partial agonist of PG receptors and produces its antagonism of PGE2 and PGF2alpha by that mechanism.     

Role of phospholipase and calmodulin inhibitors on insulin, arachidonic acid and prostaglandin E2 release

Using several experimental approaches, we have studied simultaneously the effect of glucose upon insulin, arachidonic acid and prostaglandin E2 release by rat pancreatic islets. A 16.6 mmol/l glucose concentration stimulated the release of insulin, arachidonic acid and prostaglandins. All these effects were significantly reduced either by calmodulin and phospholipase A2 inhibitors, or by the omission of calcium in the incubation medium. Phospholipase A2 inhibitors do not modify the glucose-induced net 45Ca2+ uptake by isolated islets. Our results would suggest that activation of phospholipases, particularly A2, is involved in the mechanism by which glucose stimulates insulin release. This activation increases the intracellular concentration of arachidonic acid, prostaglandins and probably phospholipid degradation products, that could act as messengers for the stimulus-secretion coupling of insulin. The calcium-calmodulin complex would take part in this effect. Conversely, the glucose-induced net calcium uptake by the islets might either be preceded by phospholipase activation or not significantly affected by the blockade of its activity.     

Failure to monoaminergic and cholinergic receptor blockers to prevent prostaglandin E2-induced luteinizing hormone release.

Receptor blocking drugs were used to determine whether adrenergic, dopaminergic, serotoninergic, or cholinergic synapses are involved in mediating the LH release induced by intraventricularly injected PGE2. Prostaglandin E2 (5mug) was injected into the 3rd ventricle (3rd V) of ovariectomized rats, and plasma LH concentrations before and after treatment were determined by radioimmunoassay. Phentolamine, 20 or 30 mug, or pronethalol, 20 mug (alpha and beta adrenergic receptor blockers, respectively) injected into the 3rd V failed to alter the elevation of plasma LH evoked by PGE2 injected into the ventricle 10 min later. Likewise, LH release following PGE2 was not changed when a dopaminergic blocker, pimozide (0.63 mg/kg, SC), was injected 2 h prior to PGE2. Two antagonists of serotonin, methysergide maleate (3 mg/kg ip) or cinanserin HC1 (1 mg/kg iv) given 2 h or 45 min before PGE2, respectively, failed to alter the action of PGE2. Atropine (100 or 250 mug) injected into the 3rd V 10 min prior to PGE2 was also ineffective in blocking the increase in plasma LH following PGE2. The results of this study indicate that the effect of PGE2 on LH release is not mediated by adrenergic, dopaminergic, serotoninergic, or cholinergic receptors. They also suggest that PGE2 is not acting trans-synaptically but probably directly on the LHRH neuron to induce the discharge of LHRH into the hypophysial portal vessels which then evokes release of LH from the adenohypophysis.     

Mechanism of prostaglandin E2-induced glucose production in rat hepatocytes

Effects of prostaglandin E2 (PGE2) on glycogenolysis were examined in rat hepatocytes. In a batch incubation system using isolated hepatocytes, PGE2 increased glucose output dose-dependently. The glycogenolytic effect of PGE2 was detected at a concentration of 10(-11) M, and 10(-8) M PGE2 elicited the maximum glucose output, which was equal to that by glucagon. PGE2 did not increase cAMP at any dose tested (10(-11)-10(-4) M). Instead, PGE2 increased the cytoplasmic free calcium concentration ([Ca2+]c). When the effect of PGE2 on [Ca2+]c was studied in aequorin-loaded cells, the effect of PGE2 on [Ca2+]c was detected at 10(-12) M, and the magnitude of the response increased in a dose-dependent manner. PGE2 increased [Ca2+]c even in the presence of 1 microM extracellular calcium, suggesting that PGE2 mobilizes calcium from an intracellular pool. In line with these observations, PGE2 increased the production of inositol trisphosphate. Compared with the action of PGE2, 16,16-dimethyl-PGE2, a PGE2 analog, was less potent in stimulating glycogenolysis. These results indicate that PGE2 stimulates glycogenolysis by activating the calcium messenger system.     

Stimulation of leptin release by arachidonic acid and prostaglandin E(2) in adipose tissue from obese humans

The purpose of this study was to examine the effect of arachidonic acid and its metabolites on leptin formation by explants of human adipose tissue over a 48-hour incubation in primary culture. We found that arachidonic acid or prostaglandin E(2) (PGE(2)) stimulated leptin release by explants of subcutaneous adipose tissue from obese humans. The stimulatory effect of arachidonic acid on leptin formation was blocked by NS-398, a cyclooxygenase-2 (COX-2) inhibitor. There was appreciable release of PGE(2) to the medium over 48 hours, and this was inhibited by 99% in the presence of 200 nmol/L dexamethasone or 5 micromol/L NS-398. The increase in PGE(2) release correlated with induction of COX-2 activity during the 48-hour incubation. The increase in COX-2 activity was blocked by 200nmol/L dexamethasone. The level of leptin mRNA at 48 hours was reduced by 28% if PGE(2) was added in the absence of dexamethasone, while in the presence of dexamethasone, the amount of leptin mRNA was enhanced by 156%. These data suggest that when upregulation of COX-2 is blocked by dexamethasone, exogenous PGE(2) enhances both leptin release and leptin mRNA accumulation by explants of human adipose tissue in primary culture.     

Inhibition of IL-1beta-dependent prostaglandin E2 release by antisense microsomal prostaglandin E synthase 1 oligonucleotides in A549 cells

The metabolism of arachidonic acid through the cyclooxygenase pathway is a highly regulated cellular process that results in the formation of PGH2. This unstable intermediate can be enzymatically metabolized to PGE2 by the actions of a microsomal 17 kDa PGE synthase (mPGES1). Treatment of A549 cells with IL-1beta for 24 h resulted in a twofold increase in mPGES1 mRNA, protein expression, and PGES specific activity. To understand the relationship between expression of mPGES1 and PGE2 formation, IL-1beta treated cells were incubated with increasing concentrations of antisense oligonucleotides (ASO) and their effects compared to cells treated with reverse sense oligonucleotides (RSO) designed against the ATG translation initiation codon of mPGES1. Incubation with ASO resulted in a 44% reduction in mRNA expression level as compared to RSO-treated cells. Microsomal preparations isolated from ASO- and RSO-treated cells were analyzed for their ability to convert PGH2 to PGE2 in the presence 2.5 mM reduced glutathione. An approximate 50% reduction (ASO: 1.8 nmol/min/mg, RSO: 3.7 nmol/min/mg) in PGES activity, protein expression by immunodetection, and extracellular PGE2 release was detected in these samples. As a control in these studies, the protein levels of COX2 and secreted IL-8 were quantified; no change in these levels was observed. These results demonstrate the direct association between mPGES1 expression, its enzymatic activity, and total PGE2 production following an inflammatory stimulus.     

Modulation of rat gastric mucosal prostaglandin E2 release by dietary linoleic acid: effects on gastric acid secretion and stress-induced mucosal damage

We studied chronic intake of diets deficient in or supplemented with linoleic acid to determine whether it affects gastric acid secretion, release of prostaglandin E2, and stress-induced lesions. For 8-10 wk rats were fed three dietary regimens supplying 3.5% (control group), 0.3%, and 10% of total calories as linoleic acid. We found that diets deficient in linoleic acid (0.3%) reduced release of prostaglandin E2 into the gastric lumen (-77%) and increased basal (+133%) and pentagastrin-stimulated acid secretion (+93%) and the area of cold restraint-induced gastric mucosal lesions (+280%), when compared with the control group. Diets supplemented with linoleic acid (10%) increased prostaglandin E2 release into the gastric lumen (+106%) and reduced basal (-44%) and pentagastrin-stimulated acid secretion (-78%) and the area of cold restraint-induced mucosal.lesions (-80%). Prevention of these lesions by the 10% linoleic acid diet was confirmed by quantitative histology. Pretreatment with indomethacin (8 mg/kg intraperitoneally) abolished the effects of the 10% linoleic acid diet on prostaglandin formation, acid secretion, and mucosal injury. We conclude that in rats chronic intake of dietary linoleic acid reduces acid secretion and prevents cold restraint-induced mucosal lesions, possibly because of augmented synthesis of endogenous prostaglandins in the gastric mucosa.     

Nitric oxide mediates norepinephrine-induced prostaglandin E2 release from the hypothalamus.

Nitric oxide (NO), formed by conversion of arginine to citrulline and NO by NO synthase, mediates relaxation of vascular smooth muscle. NO synthase has been demonstrated by immunocytochemical methods in neurons in various parts of the central nervous system including the hypothalamus. The latter finding suggested to us that NO might play a role in controlling the release of hypothalamic peptides. We have previously shown that norepinephrine mediates the release of luteinizing hormone-releasing hormone (LHRH) from LHRH terminals in the median eminence into the hypophyseal portal veins, which transport LHRH to the anterior pituitary gland to trigger release of luteinizing hormone from gonadotrophs. LHRH release from these terminals requires increased release of prostaglandin E2 (PGE2). PGE2 activates adenylate cyclase to produce cAMP, and then cAMP induces the exocytosis of LHRH secretory granules. In view of the evidence above and because of the developing evidence for the importance of NO in the central nervous system, it occurred to us that NO might be involved in this process. Consequently, we evaluated the role of NO in the release of PGE2 from medial basal hypothalamic fragments. As previously reported, norepinephrine (10 microM) increased PGE2 release from the hypothalamic fragments. The inhibitor of NO synthase NG-monomethyl-L-arginine (NMMA, 300 microM) blocked the stimulation of PGE2 release induced by norepinephrine but had no effect on the basal release of PGE2. Sodium nitroprusside (100 microM), which liberates NO, also elevated PGE2 release from the hypothalamic fragments. This elevation was not affected by NMMA, presumably because NMMA blocks enzymatic generation of NO but does not alter NO liberated by nitroprusside. When the NO liberated by nitroprusside was inactivated by hemoglobin (2 micrograms/ml), the effect of nitroprusside on PGE2 release was completely inhibited. Neither NMMA nor hemoglobin altered the basal release of PGE2, which indicates that NO is not responsible for basal PGE2 release. Addition of L-arginine (10 microM to 1 mM), the substrate for NO synthase, had no effect on basal PGE2 production. These results indicate that NO synthase is not activated in unstimulated hypothalamic fragments in vitro. The results suggest that norepinephrine activates NO synthase leading to the production of NO, which subsequently activates cyclooxygenase and results in the production of PGE2. PGE2 then activates adenylate cyclase leading to generation of increased cAMP, which induces exocytosis of secretory granules of LHRH and other neuropeptides released by PGE2. The indication that NO is essential to norepinephrine-induced release of PGE2 from hypothalamic fragments provides insight into the mechanism of LHRH release and the results open the possibility that the importance of NO to neuronal functions may be widespread in the nervous system.     

Effects of an orally administered prostaglandin analogue (16, 16-dimethyl prostaglandin E2) on human gastric secretion.

The secretory response to 16, 16-dimethyl prostaglandin E2 (DMPG) administered orally in 4 different dosages and to placebo was evaluated in healthy volunteers over a 2-hr period. During stimulation of gastric secretion by histamine, DMPG at the highest dosage (1.5 mug per kg) reduced volume by 47% and acid output by 79%. Pepsin concentration was not affected. At the same dose, DMPG inhibited basal secretion by 54% and 99% for volume and acid output, respectively. There were no side effects secondary to DMPG administration, which indicates that this compound may be useful in treating peptic ulcer disease.     

Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression

Overexpression of human cyclooxygenase 2 (COX-2) in the mammary glands of transgenic mice induces tissue-specific tumorigenic transformation. However, the molecular mechanisms involved are not yet defined. Here we show that COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland. Microvessel density increased before visible tumor growth and exponentially during tumor progression. Inhibition of prostanoid synthesis with indomethacin strongly decreased microvessel density and inhibited tumor progression. Up-regulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue was also potently inhibited by indomethacin treatment, suggesting that prostanoids released from COX-2-expressing mammary epithelial cells induce angiogenesis. G protein-coupled receptors for the major product, prostaglandin E(2) (PGE(2)) EP(1-4), are expressed during mammary gland development, and EP(1,2,4) receptors were up-regulated in tumor tissue. PGE(2) stimulated the expression angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice. Such cells are tumorigenic in nude mice; however, treatment with Celecoxib, a COX-2-specific inhibitor, reduced tumor growth and microvessel density. These results define COX-2-derived PGE(2) as a potent inducer of angiogenic switch during mammary cancer progression.     

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395

The release of ADP from platelet dense granules and its binding to platelet P2Y₁₂ receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y₁₂ has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y₁₂ receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y₁₂ inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y₁₂ with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca²⁺ influx, and phosphorylation of GSK3β-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y₁₂ with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.     

Allergen-stimulated release of prostaglandin E, F2 alpha by alveolar macrophages from patients with allergic asthma]

Bronchoalveolar lavage (BAL) was performed in healthy subjects (n = 12) and patients with bronchial asthma (n = 11). Between the two groups there was no significant difference in the total number of cells and the percentage of alveolar macrophages (AM), lymphocytes (L), neutrophils (N) and eosinophils (E) in BAL fluids. When AM were cultured in vitro and stimulated with dermatophagoides farinae (DPF) antigen, the amount of PGE released by AM was significantly increased in the asthmatics. When the asthmatics, AM were sensitized with specific IgE positive serum and stimulated with DPF, they released more PGE and PGF2 alpha than those when specific IgE negative serum was used (P < 0.01). The PGE/PGF2 alpha ratio was significantly decreased. There was positive correlation between the decreased value of PGE/PGF2 alpha and patients, MCH-PC20. The increase in the amount of PGE and PGF2 alpha released and the decrease of PGE/PGF2 alpha ratio might play an important role in the pathogenesis of allergic asthma.     

Insulin, prostaglandin E2 and glucagon release by human tissue incubated in vitro. Influence of indomethacin

In view of reports that prostaglandins influence insulin and glucagon secretion, we have studied PGE2, insulin and glucagon release from fragments (15-20 mg) of human insulinoma tissue incubated in vitro in the absence or presence of indomethacin (100 mumol/liter) an inhibitor of prostaglandin synthesis. Acid-ethanol extraction of this tissue showed the following hormonal contents : insulin : 7.17 U and glucagon 84.4 ng per g of tissue (wet weight). In the absence of indomethacin, the mean release of PGE2, insulin and glucagon into the incubation medium was 3.65 +/- 1.3 pmol, 10.5 +/- 1.2 mU and 708.4 +/- 141.8 pg in two hours (mean of 5 vials containing 2 fragments of 15-20 mg of tissue). PGE2 release was significantly inhibited in the presence of indomethacin (0.89 +/- 0.23 pmol). This effect was associated with a significantly higher insulin (16.8 +/- 1.9 mU/2 hours) and lower glucagon (176 +/- 19.7 pg/2 hours) release. These results support the view that insular tissue possesses a prostaglandin synthesis system which positively modulates glucagon secretion whereas it negatively influences insulin release.     

Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis.

Lesional epidermis of psoriasis has a probable reduction in the cyclic AMP/cyclic GMP ratio. This altered ratio may in part be responsible for the characteristic glycogen storage, rapid cell proliferation, and reduced differentiation in lesional epidermis. The concentrations of prostaglandins E2 and F2alpha, free arachidonic acid, and 12L-hydroxy-5,8,10,14-eicosatetrawnoic acid in specimens of uninvolved and involved epidermis of psoriasis were measured with deuterium-labeled carriers and multiple ion analysis. Snap frozen specimens contained: 1.4 +/- 0.4 mug/g (wet weight) of arachidonic acid in uninvolved in contrast to 36.3 +/- 16.7 mug/g in involved epidermis (P = 0.015); less than 0.05 +/- 0.01 mug/g of hydroxyeicosatetraenoic acid in uninvolved in contrast to 4.1 +/- 1.9 mug/g in involved epidermis (P = 0.015); 23.6 +/- 5.0 ng/g of prostaglandin E2 in uninvolved in contrast to 33.1 +/- 5.7 ng/g in involved epidermis (P less than 0.01); and 21.0 +/- 4.4 ng/g of prostaglandin F2alpha in uninvolved in contrast to 39.0 +/- 5.9 ng/g in involved epidermis (P less than 0.01). The arachidonic acid and hydroxyeicosatetraenoic acid levels in involved epidermis were strongly correlated (r = 0.97). The increased levels of arachidonic acid and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid in involved epidermis may have diagnostic and pathophysiological importance.     

DL-2-amino-5-phosphonopentanoic acid, a specific N-methyl-D-aspartic acid receptor antagonist, suppresses pulsatile LH release in the rat

To determine whether neuroexcitatory amino acids may play a role in generating intermittent hypothalamic GnRH release, the effect of N-methyl-D-aspartate (NMDA) receptor blockade on pulsatile LH secretion was examined in male rats. The ability of the NMDA receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), to inhibit activation of the hypothalamic-pituitary gonadotroph axis that follows peripheral administration of NMDA, was first established in intact rats. Subsequently, acutely castrated rats (n = 12) bearing venous catheters received four consecutive intravenous injections of AP5 (3.75 mg/injection/rat; approx. 13.6 mg/kg BW/injection) at 15-min intervals. Blood samples were collected at 10-min intervals for 1 h before and 2 h after initiation of AP5 treatment, and plasma LH concentrations were determined by RIA. For control purposes, norvaline, and amino acid structurally related to AP5, was administered to a second group of animals (n = 7) in a quantity (2.25 mg/injection/rat; approx. 8.2 mg/kg BW/injection) equimolar to that of the NMDA receptor antagonist. A third group of animals (n = 8) received only saline, the vehicle employed to inject AP5 and norvaline. AP5, but not norvaline, resulted in a marked suppression of pulsatile LH secretion. These findings suggest that neuroexcitatory amino acids acting at the NMDA receptor may play a physiological role in generating the intermittent mode of hypothalamic GnRH release.     

Effects of calcium ionophore, A23187, on prostaglandin E2 and renin release in dogs

Effects of calcium ionophore, A23187, on prostaglandin E2 and renin release, and the interrelationship were investigated in anesthetized dogs. PGE2 concentration in arterial and renal venous plasma was measured by radioimmunoassay, and secretion rate (pg/g-min) from the kidney was calculated. The renin secretion rate (ng/g-min) was calculated simultaneously. Intrarenal infusion of A23187 at a rate of 0.5 mg/min for 2 min resulted in a significant increase in RBF. PGE2 secretion rate increased from 61 +/- 26 to 1012 +/- 566 1222 +/- 500 and 388 +/- 125 at 5, 9 and 17 min. Renin secretion rate also increased rom 1.4 +/- 0.8 to 2.3 +/- 1.3, 5.8 +/- 2.7 and 5.9 +/- 1.6 at 5, 9 and 17 min. However, systemic administration of A23187 at a rate of 0.5 mg/min for 2 min did not alter the parameter. Thus, intrarenal infusion of A23187 affects RBF, renin release and PGE2 release directly within the kidney. In indomethacin treated dogs (5 mg/kg, i.v.) intrarenal infusion of A23187 significantly decreased RBF. The renin release with A23187 was also abolished with indomethacin as well as PGE2 release. These results suggest that calcium mobilization within the kidney plays an important role in PGE2 synthesis and release, and that the renal vasodilation and renin release are secondary effects of renal PGs synthesis and release.     

Influence of the prostaglandin E1 analogue Rioprostil on the human gastric mucosa

Ten healthy volunteers received the prostaglandin E1 analogue Rioprostil 300 micrograms b.i.d. for 1 week. Endoscopically obtained biopsies were investigated with tritiated thymidine and autoradiography to determine the rate of cell proliferation and with a texture-analyzing system to measure the intracellular amount of mucus in the epithelial cells. Rioprostil did not alter the tritiated thymidine labeling index in the antral area but did significantly depress it in the fundic area. The mucus content was unchanged in the antrum but was significantly increased in the fundus. These observations indicate that Rioprostil given orally causes an enhanced cell maturation in the fundic area as well as an increased intracellular mucus content. Rioprostil seems to have no influence on the human antral area.     

Attenuation of acetaminophen hepatitis by prostaglandin E2

Acute acetaminophen hepatitis was produced in three groups of five rats given 1600 mg/kg by gavage. The protective effect of 16,16-dimethyl prostaglandin E₂, 200 µg/kg administered subcutaneously 30 min later, was compared to the protective effect ofN-acetylcysteine 1 g/kg similarly administered. All animals were killed at 24 hr, and liver tissues were compared histologically to the damage found in acetaminophen-treated controls and untreated anatomic controls. Serum transaminase values at 24 hr exceeded 1000 units in the acetaminophen control group, averaged 658 units in the acetylcysteine treated group, and were near normal (75 units) in the prostaglandin treated group (P<0.02). Liver samples (1 cm³) were removed terminally at 24 hr. Liver damage was assessed without reference to precedent history. Histopathologically, damage was most severe in the acetaminophen control group, mainly in pericentral lobular zones. The prostaglandin-treated group showed considerably less damage, which was confined to the hepatic vein area. The acetylcysteine-treated group showed an intermediate degree of damage. We conclude that dmPGE₂, given 30 min after ingestion of acetaminophen was found to be more effective in limiting liver damage than NAC in this rat model.