High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostagladin endoperoxides and thromboxane A2

We examined platelet aggregation and serotonin release, induced by less than 60 μM arachidonic acid, using washed platelet suspensions in the absense of albumin. The concentration of arachidonic acid use did not cause platelet lysis. Platelet responses induced by less than 20 μM arachidonic acid were inhibited by aspirin, whereas those induced by above 30 μM arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although phosphatidic acid and 1,2-diacylglcerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while palmitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate / creatin phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachodinic acid, as those induced by 40 μM arachodonic acid. These results suggest that the mechanism of the actions of more than 30 μM arachodinic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A₂ and, perhaps, phosphatidic acid and 1,2-diacylglycerol.     

Announcement: VIth winter prostaglandin conference

Prostaglandin production by methylcholanthrene-transformed 3T3 mouse fibroblasts (MC5-5), human normal fibroblasts (D550), and canine kidney (MDCK) cells was inhibited by several analogues of anthracene, phenanthrene, and benzoflavone. Among the most effective inhibitors of MC5-5's prostaglandin production were anthracene (ID₅₀=1.7 μM), 9,10-diaminophenanthrene (ID₅₀=0.48 μM), and 7,8-benzoflavone (ID₅₀=0.55 μM). Under identical conditions, the ID₅₀'s for indomethacin and aspirin were 0.049 and 8.9 μM respectively. Production of radioactive prostaglandins by MC5-5 cells from tritiated phospholipids was blocked by 7,8-benzoflavone, but not the release of radioactive arachidonic acid.     

Prostaglandin biosynthesis in rabbit kidney medulla: Inhibition vs. by aspirin, indomethacin and meclofenamic acid

The non-steroidal anti-inflammatory drugs aspirin, indomethacin and meclofenamic acid were compared for their potency and duration of inhibition of prostaglandin biosynthesis in rabbit kidney medulla. Indomethacin and meclofenamic acid showed equal potency of inhibition (IC₅₀ 0.88 μM and 0.85 μM respectively) while aspirin was a much weaker inhibitor (IC₅₀ 120 μM). , indomethacin was the most powerful inhibitor (ID₅₀ 0.034 mg/kg) followed by meclofenamic acid (0.45 mg/kg) and aspirin (2.35 mg/kg).Studies on the duration of inhibition by these compounds showed the effect of indomethacin and meclofenamic acid to be completely reversed within 4–6 hours. In contrast, return of kidney prostaglandin biosynthetic activity following aspirin inhibition is very slow and significant inhibition is still present 48 hours after a single aspirin injection. The inhibitory effect of aspirin could be blocked by pretreatment with indomethacin, indicating that both drugs interact with related sites on the cyclo-oxygenase enzyme. The irreversible inhibition of the cyclo-oxygenase by aspirin as demonstrated in studies of other investigators suggests that the return of kidney prostaglandin synthetase activity after aspirin inhibition represents synthesis of new cyclo-oxygenase protein.     

Mucus glycoprotein secretion by duodenal mucosa in response to luminal arachidonic acid

The effect of luminal application of arachidonic acid on the alkaline secretion, prostaglandin generation, and mucus glycoprotein output and composition was studied in proximal and distal duodenum of conscious dogs. Surgically prepared duodenal loops were instilled in vivo for up to 2 h with saline (control) followed by various concentrations (12.5–100 μg/ml) of arachidonic acid. The experiments were conducted with and without intravenous pretreatment with indomethacin. The recovered instillates were assayed for the content of prostaglandin and HCO₃⁻, and used for the isolation of mucus glycoprotein. Exposure of duodenal mucosa to arachidonic acid led to concentration-dependent increase in the output of HCO₃⁻ and prostaglandin generation. In both cases this response was greater in the proximal duodenum. Pretreatment with indomethacin caused reduction in the basal HCO₃⁻ and prostaglandin output, and prevented the increments evoked by arachidonic acid. The proximal and distal duodenum displayed similar basal output and composition of mucus glycoprotein. Comparable increases in these glycoproteins were also obtained with arachidonic acid, the effect of which was abolished by indomethacin. Compared to basal conditions, mucus glycoproteins elaborated in response to arachidonic acid exhibited higher contents of associated lipids and covalently bound fatty acids, and contained less protein. The associated lipids of mucus glycoproteins elaborated in the presence of arachidonic acid showed enrichment in phospholipids and decrease in neutral lipids. The carbohydrate components in these glycoproteins also exhibited higher proportions of sialic acid and sulfate. The changes brought about by arachidonic acid were prevented by indomethacin pretreatment, and in both cases the glycoprotein composition returned to that obtained under basal conditions. The enrichment of mucus glycoprotein in lipids, sialic acid and sulfate in response to endogenous prostaglandin may be of significance to the function of this glycoprotein in the hostile environment of the duodenum.     

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Effects of acetaminophen on the renal inner medullary production of prostaglandin E₂ and F_2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E₂ and F_2α accumulation in media with a K_i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E₂ observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E₂ and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E₂ and prostaglandin F_2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID₅₀'s for prostaglandin (PG) E₂ synthesis in these cells were 0.1 μM for 2,6-xylenol, 5 μM for tricresol, 6 μM for -cresol, 7 μM for -cresol, 15 μM for 3,5-xylenol, 30 μM for -cresol and 100 μM for phenol. The corresponding values for aspirin and indomethacin were 4 μM and 0.02 μM, respectively.The substituted phenols also inhibited serotonin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG₂.     

Formamidines as synergists of cypermethrin in susceptible and pyrethroid resistant house flies (Diptera: Muscidae)

Mechanisms of formamidine synergism of pyrethroid insecticides were investigated in the house fly, Musca domestica L. A bioassay method was developed to show formamidine synergism of cypermethrin in adult house flies. Flies were exposed to a residue of cypermethrin with and without formamidine for 30 min and then transferred to a clean container. Mortality was recorded 24 h later. Synergism of cypermethrin occurred in flies exposed simultaneously to cypermethrin plus formamidines and in flies exposed to cypermethrin either before or after exposure to a formamidine. Synergism ranged up to 11.8-fold and was greater in susceptible than in resistant house flies. A monomeric derivative of amitraz (BTS 27271) was the most active synergist, chlordimeform was intermediate, and amitraz was least active. Synergism of cypermethrin also occurred in flies injected with octopamine and then exposed to cypermethrin, suggesting that formamidines may be acting as octopamine agonists. Measurements of the effects of formamidines on uptake of cypermethrin showed that BTS 27271 increased uptake less than chlordimeform and that amitraz had almost no effect. Both target site and behavioral effects are discussed as possible mechanisms of formamidine synergism of cypermethrin and other pyrethroids.     

Sodium transport by isolated bullfrog small intestine. Effect of Prostaglandin E1

Addition of 446 μM prostaglandin E₁ (PGE₁) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases in transmural potential difference and short-circuit current while addition of PGE₁ to the mucosal medium caused no change in the electrical parameters. Addition of 100 μM indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 μM indomethacin, serosal 60 μM PGE₁ markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330μM) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 μM indomethacin to the mucosal medium. Serosal 60 μM PGE₁ only stimulated the M (mucosa) → S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE₁ action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

Lack of covalent modification of prostaglandin synthetase (cyclo-oxygenase) by indomethacin

We have previously shown that aspirin irreversibly inhibits prostaglandin synthetase (cyclo-oxygenase) by acetylating the active site of the enzyme. By utilizing ¹⁴C-labeled indomethacin and a close analogue, we now show that indomethacin, unlike aspirin, does not covalently modify cyclo-oxygenase. Furthermore, indomethacin binding to the enzyme may be reversible since even though indomethacin can inhibit acetylation by aspirin, when enzyme inhibited by indomethacin (1 μM) is treated with 200 μM aspirin 3 times for 1 hour each, complete acetylation of cyclo-oxygenase is achieved.     

Indomethacin but not aspirin inhibits basal and stimulated lipolysis in rabbit kidney

The concurrent effect of indomethacin or aspirin on prostaglandins (PGs) biosynthesis and on cellular fatty acid efflux were compared. Studies with rabbit kidney medulla slices and with isolated perfused rabbit kidney showed a marked difference between the two non-steroidal anti-inflammatory drugs, with regard to their effects on fatty acid efflux from kidney tissue. While aspirin effect was limited to inhibition of PGs biosynthesis, indomethacin also reduced the release of free fatty acids. In medullary slices, indomethacin inhibited the Ca²⁺ stimulation of phospholipase A₂ activity and the resulting release of arachidonic and linoleic fatty acids. In the isolated perfused rabbit kidney, indomethacin inhibited the basal efflux of all fatty acids as well as the angiotensin II — induced selective release of arachidonate. Indomethacin also blunted the angiotensin II — induced temporal changes in the efflux of all other fatty acids. Neither indomethacin nor aspirin affected significantly the uptake and incorporation of exogenous (¹⁴C)-arachidonic acid into kidney total lipid fraction.Our tentative conclusion is that indomethacin inhibits basal as well as Ca²⁺ or hormone stimulated activity of kidney lipolytic enzymes. This action of indomethacin reduces the pool size of free arachidonate available for conversion to oxygenated products (both prostaglandin and non-prostaglandin types). The non-steroidal anti-inflammatory drugs can therefore be divided into two groups: a) $\text{aspirin-type compounds}$ which inhibit PGs formation only by interacting with the prostaglandin endoperoxide synthetase and b) $\text{indomethacin-type compounds}$ which inhibit PG generation by both reduction in the amount of available arachidonate and direct interaction with the enzyme.     

Biphasic effects of nonsteroidal anti-inflammatory drugs on prostaglandin production by cultured rat calvariae

We have studied the effects on bone of three structurally dissimilar non-steriodal anti-inflammatory drugs which inhibit prostaglandin cyclo-oxygenase activity (PGH synthase); indomethacin, flurbiprofen, and piroxicam. We used cultures of half calvaria from neonatal or fetal rats to measure effects on PGE₂ production, measured by radioimmunoassay. In four day neonatal rat calvaria, indomethacin inhibited PGE₂ release into the medium by 80% at 10⁻⁸ M, while flurbiprofen and piroxicam produced similar inhibition at 10⁻⁶ M. However, at 10⁻¹⁰ M, treatment with all three compounds resulted in an increase in medium PGE₂ concentration of 60 to 120%. To assess the mechanism of this effect, bones were labeled with [³H]-arachidonic acid, washed and cultured in the presence or absence of piroxicam. At 10⁻⁶ M, piroxicam inhibited production of cyclo-oxygenase products and arachidonic acid release. However, at 10⁻¹⁰ M, there was a substantial increase in labeled products, particularly PGE₂, despite a further decrease in arachidonic acid release. In 21 day fetal rat cultures, flurbiprofen was found to increase PGE₂ release both in control cultures and cultures which had been incubated with cortisol (10⁻⁸ M) to reduce endogenous arachidonic acid release and supplied with exogenous arachidonic acid (10⁻⁵ M) to provide a substrate. These results indicate that three potent inhibitors of PGH synthase can, paradoxically, increase prostaglandin production at low concentrations. The effect does not appear to be due to increased arachidonic acid release, and could be due to increased PGH synthase activity.     

Stimulation of human T-lymphocyte chemokinesis by arachidonic acid.

The migration of human T lymphocytes, assessed in modified Boyden chambers, was chemokinetically stimulated by arachidonic acid in a dose-related manner that achieved a peak level of 127 ± 34% enhancement (mean ± SD) at 8 μM arachidonic acid. The chemokinetic effect was dependent on the metabolism of the arachidonic acid by the T lymphocytes as derivatives of arachidonic acid that do not serve as prostaglandin and thromboxane precursors were without effect, while the cyclo-oxygenase inhibitors indomethacin (ID₅₀ = 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA) (ID₅₀ = 20 μM) suppressed the stimulation of migration by arachidonic acid. The cyclo-oxygenase product 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) reproduced part of the chemokinetic effect of arachidonic acid, but the lipoxygenase product 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) as well as PGE₂, PGF_2α, and thromboxane B₂ had no stimulatory activity. The ability of ETYA, but not indomethacin, to suppress the migration of unstimulated T lymphocytes suggested that a lipoxygenase metabolite of endogenous arachidonic acid contributes to the maintenance of their normal levels of spontaneous migration.     

Inhibitors of protein synthesis,puromycin and emetine,inhibit prostaglandin E2 release by skeletal muscle

Addition of 1μM puromycin or 1 μM emetine to rat soleus muscle in vitro decreases muscle prostaglandin E₂ release by 51–77%. This inhibition appears to be caused by decreased availability of endogenous arachidonic acid for prostaglandin E₂ synthesis, because neither puromycin nor emetine inhibits muscle prostaglandin E₂ production from arachidonic acid added into the incubation medium.     

Inhibition by indomethacin and aspirin of 15-hydroxy-prostaglandin dehydrogenase in vitro

15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. K_m for prostaglandin E₁ and for NAD⁺ were found to be 3.4 μM and 1.1 × 10^?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E₁ having a K_i=1.4 × 10^?4M and a K_i^′=1.6 × 10^?5M.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation

Fresh arterial tissue generates an unstable substance (prostaglandin X) which relaxes vascular smooth muscle and potently inhibits platelet aggregation. The release of prostaglandin (PG) X can be stimulated by incubation with arachidonic acid or prostaglandin endoperoxides PGG₂ or PGH₂. The basal release of PGX or the release stimulated with arachidonic acid can be inhibited by previous treatment with indomethacin or by washing the tissue with a solution containing indomethacin. The formation of PGX from prostaglandin endoperoxides PGG₂ or PGH₂ is not inhibited by indomethacin. 15-hydro-peroxy arachidonic acid (15-HPAA) inhibits the basal release of PGX as well as the release stimulated by arachidonic acid or prostaglandin endoperoxides (PGG₂ or PGH₂). Fresh arterial tissue obtained from control or indomethacin treated rabbits, when incubated with platelet rich plasma (PRP) generates PGX. This generation is inhibited by treating the tissue with 15-HPAA. A biochemical interaction between platelets and vessel wall is postulated by which platelets feed the vessel wall with prostaglandin endoperoxides which are utilized to form PGX. Formation of PGX could be the underlying mechanism which actively prevents, under normal conditions, the accumulation of platelets on the vessel wall.     

Evidence for enhanced venous smooth muscle turnover of prostaglandin-like substance in portal veins from spontaneously hypertensive rats

The sensitivity of portal veins from 14 to 18 week-old Okamoto-Aoki spontaneously hypertensive rats to prostaglandins A₂, B₂, D₂ and F_2α were enhanced whereas the sensitivity to prostaglandin E₂ was diminished when compared with responses of veins from normotensive Wistar-Kyoto rats. Inhibition of prostaglandin synthesis with both eicosotetraynoic acid (ETYA) and indomethacin (INDO) abolished the observed differences in sensitivity to prostaglandins. Synthesis of prostaglandin-like substance (with arachidonic acid as precursor) was significantly enhanced in portal veins from spontaneously hypertensive rats. Metabolism of prostaglandins E₂ and F_2α, employing the oil-immersion technique of Kalsner and Nickerson, appeared to be similar in veins from normotensive and hypertensive rats. These findings suggest that prostaglandin synthesis is enhanced in venous smooth muscle from hypertensive rats. The increased concentration of endogenous prostaglandin at the venous smooth muscle cell may modify the responses to exogenously administered prostaglandins thus accounting, in part, for the altered sensitivity to these fatty acids.     

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogeneously supplied arachidonic acid was converted mainly to PGF_2α which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF_2α in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF_2α production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

Interralation of prostaglandin endoperoxide (prostaglandin G2) and cyclic 3′,5′-adenosine monophosphate in human blood platelets

The prostaglandin endoperoxide, prostaglandin G₂, in platelet-rich plasma may produce reversible platelet aggregation without secretion, irreversible aggregation with secretion of platelet constituents inhibited by indomethacin, or the latter effects despite indomethacin, depending on the concentration of the endoperoxide. Irreversible aggregation and platelet secretion induced by prostaglandin G₂ apparently result from the action of ADP, since these responses are inhibited by 2-n-amylthio-5′-AMP (an inhibitor of the actions of ADP on platelets) and they do not occur in heparinized platelet-rich plasma. Prostaglandin G₂ lowers the platelet level of cyclic 3′,5′-AMP. Its actions are inhibited by elevation of cyclic AMP levels by prostaglandin E₁ or dibutyryl cyclic AMP or adenosine. Like malondialdehyde production induced by thrombin, ADP, or arachidonic acid, prostaglandin G₂-induced malondialdehyde production is reduced by dibutyryl cyclic AMP and prosraglandin E₁. Platelet activation by prostaglandin G₂ is enhanced by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)-adenine.The action of prostaglandin G₂ on platelets is more complex then previously reported.