Nicotinic acid inhibits thromboxane synthesis in platelets

The formation of thromboxane A₂ by phospholipase A₂ in rat platelets is inhibited by nicotinic acid. The synthesis of PGE₂ and PGF_2α is increased.Nicotinic acid inhibits collagen-induced aggregation in rat platelets.     

Interference with thyroid histogenesis by inhibitors of collagen synthesis

Histogenesis of thyroid follicles in the chick embryo begins with a penetration by cells of the mesenchymal capsule into a solid epithelial primordium. Before penetration occurs, slits containing fibrillar material form between the epithelial cells. The fibrillar material is an epithelial cell product as shown by its formation within channels that form in cultures of isolated epithelial primordia. The drugs L- azetidine-2-carboxylic acid (LACA) and alpha, alpha'-dipyridyl, which interfere with collagen synthesis, prevent the formation of fibrils in cultured epithelial primordia and in cultures of whole thyroids. Furthermore, mesenchymal cells do not invade when whole thyroid primordia are cultured in the presence of either drug. The effects of alpha, alpha'-dipyridyl are reversed by washing out the drug; the effects of LACA are reversed by incubation with equimolar or greater amounts of L-proline added to the medium along with the drug. The results are interpreted to mean that the fibrillar material is collagen of epithelial origin, that the collagen in some way plays a role in mesenchymal penetration of the epithelial primordium, and that the epithelium is responsible for the pattern of lobulation within the developing gland.     

Aspirin inhibits rat megakaryocyte thromboxane synthesis

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B₂, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB₂ and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.     

Interference with tolerance induction in vivo by inhibitors of prostaglandin synthesis

Prostaglandins (PG) have been implicated as modulators of both humoral and cellular immune responses. In order to evaluate a possible role for PG in tolerance, the effect of inhibitors of prostaglandin synthesis on tolerance induction and circumvention has been investigated. Injection of deaggregated human gamma-globulin (DHGG) into A/J mice leads to unresponsiveness to a subsequent challenge with immunogenic aggregated human gamma-globulin (AHGG). Administration of indomethacin (IM) or acetylsalicylic acid (ASA) shortly before and after DHGG injection prevents tolerance induction. PGE2 reverses the tolerance overriding effect provided by IM. IM is not able to overcome unresponsiveness when given 10 and 20 days after tolerance induction, at a time point when both T and B lymphocytes are tolerant. As previously shown, lipopolysaccharide (LPS) both inhibits the induction of tolerance to HGG and circumvents tolerant T helper cells late in tolerance when competent B cells are present. In contrast, IM is unable to circumvent T-helper cell tolerance when given at Day 60 after tolerogen, when B cells (but not T cells) are responsive. Furthermore, LPS acts as an adjuvant, B-cell mitogens, inducer of polyclonal Ig secretion, and primes mice when given with tolerogen, while IM has none of these properties. These results indicate a difference between the effects of IM and LPS on tolerance and a possible role of PG in DHGG-mediated tolerance induction.     

The relationship between thromboxane and cytosolic calcium modifiers in rat platelets.

Platelet activity is controlled, in part, by cytosolic free ionized calcium concentration ([Ca++]i). Regulation of platelet thromboxane (TXB2) synthesis may be by regulation of [Ca++]i. Dietary linoleate is a regulator of TXB2 synthesis, therefore, it may act by influencing [Ca++]i. Aspirin is a regulator of TXB2 synthesis by inhibition of cyclooxygenase; ouabain and nifedipine are regulators of [Ca++]i. This study was conducted to determine whether these affectors of TXB2 synthesis and [Ca++]i cause associated responses. Male nonobese Zucker rats were fed diets supplying 30% of energy (en%) as fat. Dietary fat was a mixture of corn oil and beef tallow to provide 3.0, 4.5, 6.0, or 7.5 en% linoleic acid, with cholesterol added to provide equal cholesterol in all diets. Rats were fed for 30 days with 6 rats/diet. Isolated rat platelets were assayed for FA composition; the percentage of linoleic acid in platelet FA rose linearly with increasing dietary linoleate (r = 0.76, P less than 0.0001). Resting and thrombin-stimulated platelet [Ca++]i and TXB2 synthesis were measured in the presence or absence of extracellular calcium and aspirin, ouabain, or nifedipine. Aspirin caused reductions in both parameters; nifedipine blocked [Ca++]i, but did not affect TXB2; ouabain increased both. Changes induced by those modifiers of TXB2 and platelet [Ca++]i caused changes that were in the same direction for both. CaCl2 caused an increase in both and the [Ca++]i was correlated with the square root of the TXB2; without CaCl2 the two were negatively correlated; aspirin, ouabain, and nifedipine treatments resulted in no significant correlations. The results suggest that there is a common modifier of [Ca++]i and TXB2 synthesis.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO2) exposure on prostacyclin (PGI2) synthesis in the rat lung and thromboxane A2 (TXA2) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO2 for 1, 3, 5, 7 and 14 days. PGI2 synthesizing activity of homogenized lung decreased. The damage of PGI2 synthesizing activity reaches its maximum at 3 days. At 14 days, PGI2 synthesizing activity returned to the normal level. The activity of PGI2 synthetase decreased significantly. The formation of lipid peroxides due to NO2 exposure may cause the depression of PGI2 synthesizing activity of lung. On the other hand, platelet TXA2 synthesizing activity increased. This increased TXA2 synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO2 exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO2 exposure. These results indicate that the depression of PGI2 synthesizing activity of lung by NO2 exposure cause an increase in TXA2 synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO2 exposure.     

Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells

Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A2. When primary cultures of human umbilical vein endothelial cells were incubated with 14C-arachidonic acid and the 14C-metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F1alpha and thromboxane B2, the degradation products of prostacyclin and thromboxane A2, respectively. Since platelets synthesize thromboxane A2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10(-5) M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F1alpha and thromboxane B2. The production of thromboxane B2 decreased in the passaged cells (207 +/- 44 pg/ml versus 65 +/- 12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F1alpha was measured in the passaged cells compared to the primary cultures (3159 +/- 356 pg/ml versus 1678 +/- 224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83 +/- 15 pg/ml versus 65 +/- 12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B2 and 6-keto-prostaglandin F1alpha decreased. In contrast, the thromboxane A2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F1alpha. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.     

Interference with S and G2 phase progression by polyamine synthesis inhibitors

The distribution of Ehrlich ascites tumor cells in the cell cycle was studied by flow cytometry following treatment with α-methyl ornithine (αMO) or methylglyoxal-bis(guanylhydrazone) (MeGAG). αMO and MeGAG are potent inhibitors of ornithine decarboxylase (putrescine synthesis) and S-adenosyl-methionine decarboxylase (spermidine and spermine synthesis), respectively. The data show that these polyamine synthesis inhibitors produce significant cell cycle perturbations resulting in an accumulation of cells in S and G2. This fact suggests that a normal polyamine complement is essential for the progression through these phases of the cell cycle.     

Effect of ethanol on thromboxane and prostacyclin synthesis by fetal platelets and umbilical artery

The effect of ethanol (10-500 mmol/l) on platelet thromboxane production and on vascular thromboxane and prostacyclin was studied in human fetal tissues. The release of thromboxane B2 (a metabolite of thromboxane A2) during thrombin-induced spontaneous aggregation of fetal platelets was inhibited by ethanol concentrations of 50 mmol/l or higher. Ethanol at concentration from 100 mmol/l also inhibited umbilical artery production of thromboxane B2 and that of 6-keto-prostaglandin F1 alpha (a metabolite of prostacyclin). However, it stimulated the conversion of exogenous arachidonic acid to thromboxane B2 in fetal platelets and to 6-keto-prostaglandin F1 alpha in the umbilical artery. This suggests that ethanol inhibits phospholipase A2, but stimulates the enzymes distal from phospholipase A2 in the prostaglandin-synthesizing enzyme cascade.     

Pharmacology of thromboxane synthetase inhibitors

Several selective and potent thromboxane synthetase inhibitors have now been described. In this paper I shall summarize their effects on platelet aggregation, endotoxin- and thrombin-induced pulmonary hypertension, the Forssman reaction-induced thrombocytopenia, and the rat tail vein bleeding time. Although any clinical utility of thromboxane synthetase inhibition remains to be demonstrated, it may be useful in conditions where vasoconstriction or vasospasm are involved, particularly where platelet activation is a contributing factor.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO₂) exposure on prostacyclin (PGIP₂) synthesis in the rat lung and thromboxane A₂ (TXA₂) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO₂ for 1, 3, 5, 7 and 14 days. PGI₂ synthesizing activity of homogenized lung decreased. The damage of PGI₂ synthesizing activity reaches its maximum at 3 days. At 14 days, PGI₂ synthesizing activity returned to the normal level. The activity of PGI₂ synthetase decreased significantly. The formation of lipid peroxides due to NO₂ exposure may cause the depression of PGI₂ synthesizing activity of lung. On the other hand, platelet TXA₂ synthesizing activity increased. This increased TXA₂ synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO₂ exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO₂ exposure. These results indicate that the depression of PGI₂ synthesizing activity lung by NO₂ exposure cause an increase in TXA₂ synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO₂ exposure.     

Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Ultra-violet radiation and 8-methoxypsoralen have actions similar to those of known inhibitors of thromboxane A2 synthesis in rat mesenteric blood vessels

In the rat mesenteric vascular bed three structurally different agents (imidazole, benzydamine and N-0164) which have been reported to be inhibitors of thromboxane (TX) A2 synthesis at certain concentrations, all have a characteristic spectrum of action. They inhibit pressor responses to nor-adrenaline and angiotensin with equal potency and the inhibition can be reversed by exogenous PGE2: they do not inhibit responses to potassium. Ultra-violet (UV) radiation has a similar spectrum of action. The main difference between the action of imidazole and that of UV radiation is that the former is rapidly reversible while the latter is not. However irradiation administered to preparations inhibited by imidazole has no irreversible effect provided that the radiation is switched off before the imidazole is removed. The imidazole protects against radiation damage suggesting that the drug may stabilize the site affected by UV light. 8-methoxypsoralen, a light sensitizing agent used in treatment of psoriasis also inhibited noradrenaline and angiotensin but not potassium responses and seemed to make the preparation more sensitive to radiation damage. It is possible that UV radiation and 8-methoxypsoralen may inhibit TXA2 synthesis but this requires confirmation by direct methods.     

Effects of inhibitors of thromboxane synthesis on reactions of the cat bronchus in situ to prostaglandins

Cats were anesthetized by chloralose, relaxed by suxamethonium, and ventilated. The influence of 3 inhibitors of thromboxane synthesis, N.0096 (±α-benzyl-α-p-chlorobenzyl-4-hydroxyacetophenon phenylhydrogen-phosphonate), imidazole and dipyridamole were studied on bronchial reactions to prostaglandins E₂ and F₂α and arachidonic acid. During a bronchoconstriction maintained by infusion of 5-HT, N.0096 and imidazole dose-dependently potentiated the intensity and duration of the bronchodilator effect of PGE₂. This effect was maximal at a total dose of 17 mg/kg N.0096 or 25–50 mg/kg imidazole, but decreased after higher doses which are supposed to inhibit cyclo-oxygenase also. Bronchoconstrictor reactions to single injections of arachidonic acid were inhibited by N.0096 in the same dose range. Reactions to PGF_2α were unaltered by both drugs. Dipyridamole was devoid of a comparable activity.     

Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.     

Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain.

—Certain of the sulphur containing amino acids have been associated with synaptic transmission in the central nervous system. The enzymes involved in the synthesis of these putative neurotransmitter or modulator compounds have a different subcellular distribution in rat brain from those enzymes that catalyse the synthesis of other compounds in this pathway. Methionine adenosyltransferase and 5-methyltetrahydrofolate-homocysteine methyltransferase catalyse reactions that maintain the methylation functions of the pathway and are found in soluble fractions. Cystathionine β-synthase, cystathionase, cysteine dioxygenase and cysteine sulphinic acid decarboxylase catalyse the synthesis of those sulphur-containing amino acids implicated in neurotransmitter functions and these enzymes have both paniculate and soluble components. Serine hydroxymethyltransferase, which also has a particulate fraction in brain, is responsible for the synthesis of the neurotransmitter glycine, in addition to its role in the methionine-related metabolism of folate.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.