Production of Hyperalgesic Prostaglandins by Sympathetic Postganglionic Neurons

Prostaglandin E2 and prostacyclin (prostaglandin I2) produce hyperalgesia in animals and humans. Because there is evidence that prostaglandins contribute to pain maintained by sympathetic nervous system activity, we evaluated whether sympathetic postganglionic neurons synthesize these hyperalgesic prostaglandins, and whether production of prostaglandins by these neurons can contribute to sensitization of primary afferent nociceptors. Intradermal injection of arachidonic acid but not linoleic acid, in the rat hindpaw, produces a decrease in mechanical nociceptive threshold. This hyperalgesic effect is prevented by indomethacin, an inhibitor of prostaglandin synthesis or by prior surgical removal of the lumbar sympathetic chain. To test the hypothesis that sympathetic postganglionic neurons are the source of prostaglandins, we measured production of prostaglandin E2 and 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) by homogenates of adult rat sympathetic postganglionic neurons from superior cervical ganglia. These homogenates produced significant amounts of prostaglandin E2 and 6-keto-prostaglandin F1 alpha, and most of this production is eliminated by neonatal administration of 6-hydroxydopamine which selectively destroys sympathetic postganglionic neurons. These results demonstrate that sympathetic postganglionic neurons produce prostaglandins, and supports further the hypothesis that the release of prostaglandins from sympathetic postganglionic neurons contributes to the hyperalgesia associated with sympathetically maintained pain.     

Reduction of the hyperthermic effect of prostaglandin E2 by cholinomimetics, monoamines and calcium ions]

It was shown in experiments on unanesthetized rats with prostaglandin E2 (PGE2) hyperthermia, not preventable by aspirin that intraventricular (into the lateral ventricle) injections of arecoline, noradrenaline, 5-hydroxytryptamine (5-HT), histamine, and calcium ions and intraperitoneal eserine injection were capable of decreasing body temperature. PGE2 hyperthermia was not prevented by aspirin, but it was reduced by eserine. After the administration of arecoline and nicotine into the third ventricle of unanesthetized rabbits with PGE2 hyperthermia body temperature decreased as well. The effect of arecoline and 5-HT was reproducible in the same animals. The data are suggestive of the existence in the heat loss centre of mechanisms including cholinergic neurons whose activity was not completely suppressed by PGE2.     

On the synthesis of prostaglandins by human gastric mucosa and its modification by drugs.

1. Specific radioimmunoassays for the prostaglandins E2, A2 and F2alpha were used to study the synthesis of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric corpus mucosa. 2. Both prostaglandin E2 and prostaglandin F2alpha were found to be synthesized from arachidonic acid by themicrosomal fraction of human gastric mucosa. The synthesis of prostaglandin E2 exceeded that of prostagladin F2alpha by a factor of about 10. 3. Synthesis of prostaglandin A2 or prostaglandin B2 was not observed under the same incubation conditions. 4. Indometacin effectively inhibited synthesis of both prostaglandin E2 (ID50 4.2 microng/ml) and prostaglandin F2alpha (ID50 1.8 microng/ml) by human gastric mucosa, while paracetamol even in a concentration of 310 microng/ml did not influence prostaglandin synthesis. The anti-ulcer agent carbenoxolone, which has been shown to inhibit prostaglandin inactivation, at the same concentration only slightly inhibited (about 20%) prostaglandin synthesis. 5. The results support the hypothesis that the gastro-intestinal effects or side effects of several drugs are mediated by an influence on the enzymes of prostaglandin synthesis or inactivation.     

Effect of organic sulfur compounds on the chemical and enzymatic transformations of prostaglandin endoperoxide H2.

The effects of several sulfur organic compounds on the enzymatic and non-enzymatic transformations of prostaglandin endoperoxide H2 to prostaglandins were studied. Mercaptoethanol, methional alpha-lipoic acid and dimercaptopropanol increased the chemical (i.e. non-enzymatic) reduction of prostaglandin H2 to prostaglandin F2alpha but except for alpha-lipoic acid, had no effect on the enzymatic conversion of prostaglandin H2 to prostaglandin. In contrast, reduced glutathione showed no effect on the chemical conversion of prostaglandin H2, but exerted a marked and specific stimulation on the enzymatic isomerization of prostaglandin H2 to prostaglandin E2. This specific effect of gluthione may serve to regulate the overall intracellular activity of prostaglandin synthetase as well as the particular ratio of prostaglandins produced.     

Effects of prostaglandin E2, indomethacin and adenosine deaminase on basal and insulin-stimulated glucose metabolism in human adipocytes

The effects of prostaglandin E2 were studied on glucose metabolism (3-O-methylglucose transport, CO2 production and lipogenesis) in human adipocytes. Initially, the effects of endogenously produced adenosine and prostaglandins were indirectly demonstrated by using adenosine deaminase and indomethacin in the incubations. From these studies it was found that adenosine deaminase (5 micrograms/ml) had a pronounced effect on adipocyte glucose metabolism in vitro. In the basal (nonhormonal-stimulated) state, glucose transport, CO2 production and lipogenesis were inhibited by about 30% (P less than 0.05). Furthermore, adenosine deaminase significantly inhibited the isoproterenol- and insulin-stimulated CO2 production and lipogenesis (P less than 0.01). Indomethacin (50 microM) had a consistently inhibitory effect on the insulin-stimulated CO2 production (P less than 0.05), whereas indomethacin had no significant effects on basal or isoproterenol-stimulated glucose metabolism. In contrast to the relatively minor effect of endogenous prostaglandins, the addition of exogenous prostaglandin E2 significantly stimulated the glucose transport, glucose oxidation and lipogenesis in human adipocytes, especially in the presence of adenosine deaminase. Half-maximal stimulation was obtained at prostaglandin E2 concentrations of 2.2, 0.8 and 0.8 nM, respectively. The effect of prostaglandin E2 was specific, since the structurally related prostaglandin, prostaglandin F2 alpha, had practically no effect on glucose metabolism. The maximal effect of prostaglandin E2 (1 microM) on glucose metabolism was 30-35% of the maximal insulin (1 nM) effect. When insulin and prostaglandin E2 were added together, the effect of prostaglandin E2 on glucose metabolism was additive at all insulin concentrations tested.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E2 and F2 alpha, thromboxane B2 and 6-keto-prostaglandin F1 alpha was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E2 accounted for 44% and 6-keto-prostaglandin F1 alpha for 28% of all prostaglandin release, and the rank order of prostaglandin release was E2 greater than 6-keto-prostaglandin F1 alpha greater than thromboxane B2 greater than prostaglandin F2 alpha. Hypoxia had no significant effect on quantitative prostaglandin release, but the ratio of prostaglandin E2 to prostaglandin F2 alpha was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F1 alpha was significantly decreased, as was the ratio of 6-keto-prostaglandin F1 alpha to thromboxane B2. Also the ratio of the vasodilating prostaglandins (E2, 6-keto-prostaglandin F1 alpha) to the vasoconstricting prostaglandins (thromboxane B2, prostaglandin F2 alpha) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparations. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

Sensitization of alveolar macrophages to lipopolysaccharide-induced prostaglandin synthesis by exogenous prostaglandins

Rabbit alveolar macrophages were found to produce extraordinary amounts of prostaglandin E2 and F2 alpha with the stimulation of lipopolysaccharide or lipid A. Exogenous prostaglandin E2 greatly enhanced the lipopolysaccharide action on rabbit alveolar macrophages for the induction of prostaglandin F2 alpha release (3-5 fold), while prostaglandin E2 alone did not cause any effect. The enhancement expressed was especially strong when prostaglandin E2 was administered to the cells simultaneously with lipopolysaccharide. The effect of prostaglandin E2 was observed neither with a nonstimulating dose of lipopolysaccharide nor with a stimulating dose of zymosan. This phenomenon was even more pronounced when prostaglandin I2 was used instead of prostaglandin E2, while no sensitization was demonstrated by prostaglandin F2 alpha. These observations suggest that prostaglandins can modulate the activation of the cyclooxygenase pathway of arachidonate metabolism in the activated macrophages by lipopolysaccharide.     

Stimulation of prolactin synthesis and of adenosine 3'':5''-cyclic phosphate formation by prostaglandins and thyroliberin in cultured rat pituitary cells.

1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.     

Uterine vein prostaglandin levels in late pregnant dogs.

We studied the uterine venous plasma concentrations of prostaglandins E2, F2 alpha, 15 keto 13,14 dihydro E2 and 15 keto 13,14 dihydro F2 alpha in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E2 and F2 alpha in pregnancy in vivo. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E2 and 15 keto 13,14 dihydro E2 were 1.35 +/- .27 ng/ml and 1.89 +/- .37 ng/ml, respectively; however, we could not find any prostaglandin F2 alpha and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F2 alpha and E2 from endoperoxides, prostaglandin F2 alpha production in vivo must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E2 is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F2 alpha does not appear to play a role at this stage of pregnancy.     

Effect of prostaglandins and their analogues on hormone-stimulated glycogenolysis in primary cultures of rat hepatocytes

Hepatocytes were isolated by collagenase perfusion method from adult male rats, cultured and then prelabeled with [14C]glucose. The [14C]glycogen-labeled cells were used in experiments for effect of prostaglandins on hormone-stimulated glycogenolysis. Prostaglandin E1, prostaglandin E2 and 16,16-dimethylprostaglandin E2, but not prostaglandin D2 or prostaglandin F2 alpha, inhibited glycogenolysis stimulated by glucagon, epinephrine, isoproterenol (beta-adrenergic agonist) or epinephrine in the presence of propranolol (beta-antagonist) in primary cultured hepatocytes. The inhibitory effects on day 2 of cultures were approx. twice those on day 1. Dimethylprostaglandin E2 (10(-6)M) caused 60-70% inhibitions of the stimulations by these substances. In the case of the stimulation by glucagon, the inhibition further increased by 80-100% on day 3 of culture. Prostaglandin E1 and prostaglandin E2 caused less inhibition than dimethylprostaglandin E2 of all these stimulations. Dinorprostaglandin E1 (9 alpha,13-dihydroxy-7-ketodinorprost-11-enoic acid), which is a hepatocyte-metabolite of prostaglandin E1 and prostaglandin E2, and arachidonic acid did not have any inhibitory effects. These data indicate that the E series of prostaglandins may function as the regulation of hepatic glycogenolysis stimulated by epinephrine and glucagon, and that their rapid degradation system may contribute to the modulation of the action in liver.     

Metabolism of prostaglandins, prostaglandin analogs and thromboxane B2 by lung and liver microsomes from pregnant rabbits

Liver microsomes from pregnant rabbits converted prostaglandins F2 alpha, E1, and E2 to their 20-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E1 and E2 were also reduced to prostaglandins F1 alpha and F2 alpha, respectively, and prostaglandin E1 was isomerized to 8-isoprostaglandin E1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B2 and a number of prostaglandin F2 alpha analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin E2 approximately prostaglandin F2 alpha approximately 16,16-dimethylprostaglandin F2 alpha approximately 13,14-didehydroprostaglandin F2 alpha greater than thromboxane B2 greater than 15-methylprostaglandin F2 alpha approximately 17-phenyl-18,19,-20-trinorprostaglandin F2 alpha approximately ent-13,14-didehydro-15-epiprostaglandin F2 alpha. Similar results were obtained with liver microsomes except that thromboxane B2 was a relatively poorer substrate for hydroxylation.     

Effect of trihydroxyoctadecadienoic acids on the blood content of prostaglandins E2 and F2 alpha and 5-hydroxyeicosatetraenoic acid in rats with alloxan diabetes

It has been demonstrated that the level of prostaglandin F2 alpha and 5-hydroxyeicosatetraenoic acid (5-HETE) in the serum of alloxan-diabetic rats is reduced by 85% and 25%, respectively, whereas that of prostaglandin E2 is increased by 34%. The administration of trihydroxyoctadecadienoic acids, that have a hypoglycemic effect, to diabetic animals brings about a rise in the level of prostaglandins F2 alpha, E2 and 5-HETE by 33%, 64% and 279%, respectively, as compared to the control.     

Prostaglandin receptors on human platelets. Structure-activity relationships of stimulatory prostaglandins.

1. Synthetic analogues of prostaglandins E2 or F2a (monocyclic bisenoic prostaglandins), like the endogenous prostaglandin endoperoxides (prostaglandins G2 and H2) from platelets, and like synthetic analogues of prostaglandin H2 (bicyclic bisenoic prostaglandins), can induce aggregation of human platelets, although prostaglandins E2 and F2a themselves are inactive. 2. All the prostanoid compounds that induce platelet aggregation release 5-hydroxytryptamine from platelet dense bodies, but do not release beta-N-acetylglucosaminidase from lysosomal granules. Arachidonic acid evokes a similar response. 3. All endoperoxide analogues tested (bicyclic compounds) were powerful platelet stimulants, and all active compounds (whether mono- or bi-cyclid) apparently acted via the same receptor as the endogenous prostaglandin endoperoxides. 4. The nature and stereospecificity of substituents at positions 11 and 15 (or 16) on prostaglandin E2 are critical determinants for platelet-stimulating activity: deoxy substitution at position 11 plus methylation at position 15 (or 16) produces a potent stimulant, particularly if the groups around C-15 are in the S configuration. 5. The effects of these structural modifications are apparently due to, at least in part, a change in side-chain conformation.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

The prostacyclin analogue iloprost and prostaglandin E1 suppress sterol synthesis in freshly isolated human mononuclear leukocytes

The effects of the stable prostacyclin analogue iloprost, prostaglandin E1 and prostaglandin F2 alpha on sterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for 6 h in a medium containing lipid-depleted serum led to a 3-fold rise in the rate of sterol synthesis from [14C]acetate or tritiated water. Iloprost and prostaglandin E1 added in increasing concentrations at zero time resulted in an inhibition of the synthesis of sterols, the suppression being 50 and 55% at a concentration of 1 mumol/1, respectively. Both prostaglandins yielded a sigmoidal log dose-effect curve. In contrast, prostaglandin F2 alpha had no influence on sterol synthesis up to a concentration of 1 mumol/1. The action of the prostacyclin analogue and prostaglandin E1 on the relative rate of sterol synthesis was not immediate, since the prostaglandins had no effect when given at 6 h to the incubation medium, and the incorporation of [14C]acetate into sterols was measured thereafter. The results suggest that prostacyclin and prostaglandin E1 affect cholesterol synthesis and therefore may play a role in the regulation of cellular cholesterol homeostasis and in the development of atherosclerosis.     

Mechanism of prostaglandin biosynthesis in rabbit kidney medulla. A rate-limiting step and the differential stimulatory actions of L-adrenaline and glutathione.

Microsomal prostaglandin synthase (EC 1.14.99.1) from rabbit kidney medulla was assayed with [5,6,8,9,11,12,14,15-3H]-and [1-14C]-arachidonic acid as the substrate. The ratios of prostaglandin F2 alpha to prostaglandin E2 and to prostaglandin D2 were determined by both 3H and 14C labelling. When 3H was used as a label the ratios were much higher than with 14C labelling indicating that the removal of hydrogen at C-9 or C-11 was the rate-limiting step in the biosynthesis of prostaglandin E2 or prostaglandin D2. This finding shows that the octatritiated arachidonic acid is not the appropriate substrate marker for studying the regulation of the synthesis of different prostaglandins by various agents. When the enzyme assay was carried out in the presence of SnCL2, which was capable of accumulating exclusively prostaglandin F2alpha at the expenses of prostaglandin E2 and prostaglandin D2, the addition of L-adrenaline to the microsomal fraction either alone or with reduced glutathione equally stimulated the formation of prostaglandin F2alpha, whereas the addition of reduced glutathione to the microsomal fraction either alone or with L-adrenaline produced no additional effect. These results suggest that endoperoxide is formed as the common intermediate for the biosynthesis of three different prostaglandins in rabbit kidney medulla, and that L-adrenaline stimulates the synthesis of endoperoxide, whereas reduced glutathione facilitates the formation of prostaglandins from endoperoxide.     

Prostaglandins F2alpha and E content and uptake of Ca2+ in the myometrium during different functional states

It is shown that the amount of prostaglandins F2alpha and E in myometrium of female rabbits and a woman decreases in the process of pregnancy and increases during delivery as compared to the control. The 10(-6)M concentration of prostaglandin F2alpha evokes an intensive Ca2+ uptake by myometrium strips both in normal and in pregnant animals but has no effect on the Mg2+, Ca2+-ATPase activity of sarcolemma vesicles. The Ca2+ uptake by the myometrium strips is not affected by prostaglandin F2alpha in the presence of NaF and N-ethylmaleimide inhibiting the ATP-dependent transport of Ca2+.     

Platelet activating factor-mediated leukotriene biosynthesis in rat lungs: effect of prostaglandins E1 and F1 alpha

The effect of prostaglandins E1 and F1 alpha on peptidoleukotriene biosynthesis/release from rat chopped lung stimulated with platelet activating factor was studied. Prostaglandin E1, known to stimulate adenylate cyclase in airways, inhibited the biosynthesis of leukotrienes C4, D4 and E4 and total peptidoleukotrienes whereas prostaglandin F1 alpha, which has no effect on adenylate cyclase, did not exert any effect on total peptidoleukotriene release, though a small inhibition was found for leukotriene D4. Cyclic AMP itself inhibited peptidoleukotriene release from platelet activating factor-stimulated lung, suggesting that the effect of prostaglandin E1 is mediated by cyclic AMP.     

Identification of metabolites from peroxisomal beta-oxidation of prostaglandins

We have recently shown that isolated rat liver peroxisomes can chain-shorten prostaglandin F2 alpha and prostaglandin E2 to tetranor-metabolites. In the present report dinor-metabolites of these two prostaglandins were also identified, suggesting that the peroxisomal chain-shortening reaction of prostaglandins is a beta-oxidation reaction. Furthermore, an intermediate containing an extra double bond was isolated from incubates of prostaglandin F2 alpha with peroxisomes. This intermediate was tentatively assigned the structure 2,3-dehydroprostaglandin F2 alpha. Prostaglandin E1 and a major circulating prostaglandin F2 alpha metabolite were also metabolized to chain-shortened products by peroxisomes. The accumulation of the 2,3-dehydro-metabolite and the dinor-metabolites suggest that the peroxisomal beta-oxidation sequence is not tightly coupled, in contrast to mitochondrial fatty acid oxidation.     

Prostaglandins and steroidogenesis by isolated rabbit ovarian follicles

Prostaglandins of the F and E series at concentrations from 1 to 100 microgram/ml had no effect on steroidogenesis by isolated rabbit follicles. Indomethacin and 7-oxa-13-prostynoic acid at doses lower than 100 microgram/ml failed to prevent the LH-induced increase in testosterone accumulation by follicles. At 1 mg/ml these inhibitors prevented the LH effects. Prostaglandin E2 and F2alpha had no effect on testosterone accumulation. However, prostaglandin E2 seemed to enhance the LH-induced accumulation of androstenedione and progesterone by the follicles. These data suggest that prostaglandins play a minor role in steroidogenesis by isolated rabbit ovarian follicles.