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.     

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.     

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on in vitro studies on ram seminal vesicles and in vivo drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers. Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB2 were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart). While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.     

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on studies on ram seminal vesicles and drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers.Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB₂ were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart).While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.     

Inhibition of the oxygen-induced contraction of the isolated human umbilical artery by indomethacin, flurbiprofen, aspirin and drugs modifying Ca2+ disposition

In this study we have quantified the potency of three cyclo-oxygenase inhibitors, indomethacin, flurbiprofen and aspirin to reduce the vasoconstriction of isolated strips of human umbilical artery (HUA) to changing the oxygen-tension (PO2) of the bathing medium. The inhibitory potencies (IC50) of indomethacin and flurbiprofen were similar (IC50 = 2.1 x 10(-9) M and 7.6 x 10(-9) M respectively) while aspirin (IC50 = 2.5 x 10(-5) M) was approximately 12,000 fold less potent than indomethacin. At the physiological PO2 of 15 mmHg the isolated HUA was found to have an inherent cyclo-oxygenase induced tone since either reducing the PO2 from 15 to OmmHg or adding cyclo-oxygenase inhibitors caused vasorelaxation. O2-induced contractions were partly dependent on extracellular Ca2+: nifedipine and Bay K 8644 inhibited and enhanced oxygen-induced contractions, respectively, but did not have a significant effect on calcium-dependent 5-hydroxy-tryptamine-induced (5-HT) contractions. Therefore cyclo-oxygenase products and 5-hydroxytryptamine-induced contractions of the HUA may both utilise extracellular calcium but through different processes.     

Effects of aspirin and indomethacin on cerebral circulation in the conscious rat: Evidence for a physiological role of endogenous prostaglandins

The purpose of this work was to evaluate the effects of equipotent doses of two different inhibitors of cyclo-oxygenase, indomethacin and aspirin, on cerebral blood flow and cerebral vascular resistances in the conscious undisturbed rat, using the reference sample radioactive microsphere method. We found that both, aspirin (50 mg/kg) and indomethacin (5 mg/kg) at 3, 15 and 60 min after their intravenous administration, increased cerebral vascular resistances and decreased cerebral blood flow to a similar extent. Both drugs completely abolished the hypotensive effect of 5 mg/kg i.v. arachidonic acid and they did not change arterial PO₂, PCO₂ or pH values. We conclude that the pharmacological inhibition of cyclooxygenase in the conscious undisturbed rat leads to a cerebral vasoconstriction and consequently to a decrease in cerebral blood flow. Our results evidence that prostaglandins are a physiological factor that actively contributes to the maintenance of cerebral circulation.     

Mobilization of arachidonic acid in collagen-stimulated human platelets.

Stimulation of platelets with collagen results in the mobilization of arachidonic acid (AA) from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). In this study the effect of aspirin, indomethacin, BW755C and prostaglandin H2 (PGH2) on labelled AA release in response to varied concentrations of collagen was investigated. Our results indicate that aspirin (0.56 mM) and indomethacin (5.6 microM) not only inhibited the collagen-mediated formation of cyclo-oxygenase metabolites, but also caused a significant reduction in the accumulation of free labelled AA and 12-hydroxyeicosatetraenoic acid (12-HETE) (21-64%). Aspirin and indomethacin also inhibited the release of [3H]AA from PC (37-75%) and PI (33-63%). The inhibition of AA release caused by aspirin was reversed partially by PGH2 (1 microM). In contrast, a smaller/no inhibition of collagen-stimulated labelled AA and 12-HETE accumulation (0-11%) and of collagen-stimulated AA loss from PC and PI was observed in the presence of BW755C. The results obtained in the presence of aspirin, indomethacin and BW755C at lower concentrations of collagen further demonstrate that AA release from PI (45-61% inhibition at 10 micrograms of collagen), but not from PC, was affected by the inhibition of cyclo-oxygenase. The results obtained on the effect of PGH2 further support that deacylation of phospholipids occurs independently of cyclo-oxygenase metabolites, particularly at higher concentrations of collagen. These results also demonstrate that aspirin and indomethacin, but not BW755C, cause a direct inhibition of collagen-induced [3H]AA liberation from PC as well as from PI. We also conclude that the diacylglycerol lipase pathway is a minor, but important, route for AA release from PI in collagen-stimulated human platelets. The mechanisms underlying the regulation of AA release by collagen in the absence of cyclo-oxygenase metabolites are not clear.     

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 micronM) is treated with 200 micronM aspirin 3 times for 1 hour each, complete acetylation of cyclo-oxygenase is achieved.     

Salicylic acid blocks indomethacin-induced cyclooxygenase inhibition and lesion formation in rat gastric mucosa

Salicylic acid has been shown to decrease gastric mucosal lesions induced by indomethacin in the rat. In vitro, it has also been shown to counteract the inhibitory effect of indomethacin and aspirin on the cyclooxygenase enzyme system in seminal vesicle microsomes and in platelets and vascular tissue. The hypothesis that the mechanism of salicylic acid "protection" against indomethacin-induced gastric lesions involves interference with indomethacin-induced mucosal cyclooxygenase inhibition was tested. Male, fasted rats were treated with intragastric salicylic acid in doses of 50, 100, 200, 300, or 400 mg/kg concomitantly with a sc injection of 20 mg/kg of indomethacin. Gastric mucosal lesions and mucosal cyclooxygenase activity (as measured by ex vivo prostaglandin F2 alpha synthesis) were examined 3 hr later. Intragastric salicylic acid, 200-400 mg/kg, significantly reduced indomethacin-induced lesion formation, while counteracting significantly indomethacin inhibition of prostaglandin synthesis. Salicylic acid alone did not significantly change cyclooxygenase activity. It is concluded that topical salicylic acid can decrease indomethacin-induced gastric mucosal lesion in the rat, in part, by counteracting the inhibitory effect of indomethacin at the cyclooxygenase level.     

Do endogenous prostaglandins modulate noradrenergic transmission in the rat isolated perfused vas deferens?

The effects of the prostaglandin synthetase inhibitors aspirin, indomethacin and meclofenamic acid have been studied on the response of the rat isolated perfused vas deferens. None of these drugs, up to a concentration of 5 x 10(-5) M affected either phase of the biphasic constrictor response to 30 s periods of field stimulation. In the same preparations an inhibition of the response to field stimulation was seen in the presence of prostaglandin E1 at concentrations of 1 to 5 ng ml-1. All three prostaglandin synthetase inhibitors, at 5 x 10(-5) M, caused significant reduction of prostaglandin biosynthesis by homogenates of rat vas deferens. It is, therefore, suggested that stimuli which activate directly the noradrenergic nerves in the rat vas do not activate simultaneously a release of endogenous prostaglandins.     

Stimulation of adenosine 3':5'-monophosphate formation by prostaglandins in human astrocytoma cells. Inhibition by nonsteroidal anti-inflammatory agents.

Prostaglandins (PG) of the E series and catecholamines stimulate adenosine 3':5'-monophosphate (cAMP) formation in human astrocytoma cells (1321N1). These two classes of effectors activated adenylate cyclase upon interaction with different receptor systems. No evidence for a mediatory role for PG in the action of catecholamines was found. PG interacted with 1321N1 cells with an order of potency of PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. The effect of combinations of the various PG indicated that all efficacious PG interacted with a common receptor. 7-Oxa-13-prostynoic acid and indomethacin were shown to be competitive inhibitors of the effect of PGE1 with Ki values of 4 and 150 micron, respectively. These two compounds did not inhibit the effect of isoproterenol. Polyphloretin phosphate caused a complex pattern of inhibition of the effects of PGE1 and at higher concentrations also inhibited the effects of isoproterenol. The mefenamate class of nonsteroidal anti-inflammatory agents was found to inhibit the effects of PGE1 with a potency order of meclofenamic acid greater than flufenamic acid = mefenamic acid. The inhibitory action of meclofenamic acid was complex involving specific, but partial, insurmountable antagonism of PGE1 as well as competitive inhibition of PGE1 effects. At higher concentrations of meclofenamic acid a nonspecific inhibition of the effects of both PGE1 and isoproterenol was observed. These studies suggest that the inhibition by nonsteroidal anti-inflammatory agents of the physiological effects of PGE1 in animals may occur, at least in part, at the level of adenylate cyclase. The possibility that multiple classes of adenylate cyclase-linked PGE receptors might exist in nature is discussed.     

Endogenous inhibition of arachidonic acid metabolism in the endometrium of the sheep

Arachidonic acid is metabolised via the cyclo-oxygenase pathway to several biologically active metabolites. These metabolites control important reproductive functions like luteolysis of the corpus luteum. The metabolism of arachidonic acid was studied by the enzymatic conversion of [1-14C]-labelled arachidonic acid in sheep endometrial tissue. The inhibitory capacity of sheep endometrial tissue was measured by the enzymatic conversion of [1-14C]-arachidonic acid by sheep seminal vesicular gland microsomes. Endometrial microsomes converted arachidonic acid into different prostaglandins and monohydroxy acids but at a low rate. A factor(s) inhibiting both prostaglandin and monohydroxy acid synthesis was found in both the microsomal and cytosolic fractions of endometrial tissue. A very high inhibitory potency of prostaglandin and monohydroxy acid synthesis, calculated as IC50 values, was found in cytosolic fractions. For comparison IC50 values of indomethacin, mefenamic acid, carprofen and acetylsalicylic acid were also calculated in this in vitro system. These data indicate that both prostaglandin and monohydroxy acid synthesizing capacities and an inhibitory factor(s) are present in sheep endometrium and possibly regulate arachidonic acid metabolism in this tissue.     

Pyrrolidine inhibitors of human cytosolic phospholipase A2. Part 2: synthesis of potent and crystallized 4-triphenylmethylthio derivative 'pyrrophenone'

We synthesized a potent and crystallized human cytosolic phospholipase A2alpha inhibitor, pyrrophenone (6) which inhibits the isolated enzyme with an IC50 value of 4.2 nM. Pyrrophenone shows potent inhibition of arachidonic acid release, prostaglandin E2, thromboxane B2, and leukotriene B4 formation in human whole blood. The magnitudes of prostaglandin E2 and thromboxane B2 inhibition are the same as those of indomethacin.     

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.     

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.     

Attenuation of hypotensive effect of propranolol and thiazide diuretics by indomethacin.

The effects of 100 mg indomethacin daily for three weeks on blood pressure and urinary excretion of prostaglandin F2 alpha were studied in a double-blind, placebo-controlled comparison of two groups of patients with essential hypertension, eight receiving propranolol and seven thiazide diuretics. Compared with placebo, adding indomethacin to the patients'' established antihypertensive treatment increased blood pressure by 14/5 Hg supine and 16/9 mm Hg erect in the patients receiving propranolol, and by 13/9 mm Hg supine and 16/9 mm Hg erect in the patients receiving thiazide diuretics (all p less than or equal to 0.05). The excretion of the major urinary metabolite of prostaglandin F2 alpha was reduced by 67% in the propranolol-treated patients and by 57% in those receiving a thiazide diuretic. Body weight increased by 0 . 8 kg (propranolol) and 1 . 1 kg (thiazide diuretic) when indomethacin was given, but there were no significant changes in creatinine clearance, urinary sodium excretion, or packed cell volume in either treatment group. These results suggest that products formed by the arachidonic acid cyclo-oxygenase contribute to the regulation of blood pressure during treatment with both propranolol and thiazide diuretics. Inhibition of the cyclo-oxygenase with indomethacin partially antagonises the hypotensive effect of these drugs.     

Aspirin-like drugs inhibit arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in rat neutrophils from carrageenan pleural exudates

Aspirin-like drugs inhibit the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase in rat neutrophils from carrageenan pleural exudates. These non-steroidal anti-inflammatory drugs inhibit the formation of 11-hydroxy- and 15-hydroxy-eicosatetraenoic acid (HETE) as well as prostaglandins. In addition, the concentration- and time-dependent irreversible inhibition of lipoxygenase by aspirin and indomethacin parallels closely the patterns observed for inhibition of cyclo-oxygenase. The results suggest that some common steps may exist for the synthesis of HETE and prostaglandins from arachidonic acid in rat neutrophils. The ability of aspirin-like drugs to inhibit the formation of the chemotactic hydroxy-fatty acids may contribute to their anti-inflammatory activity.     

The interaction between lipid peroxidation and prostaglandin synthesis in rabbit kidney-medulla slices.

Lipid peroxidation induced by ascorbic acid and Fe2+ was inhibited by mepacrine (phospholipase A2 inhibitor) and aspirin (prostaglandin cyclo-oxygenase inhibitor) in rabbit kidney-medulla slices. Moreover, ascorbic acid and Fe2+ potentiated the inhibitory effect on prostaglandin E2 formation by mepacrine, but they had no influence on prostaglandin E2 production decreased by aspirin. Lipid peroxidation induced by ascorbic acid and Fe2+ appears to be affecting the activity of prostaglandin endoperoxide synthase. These results suggest that lipid peroxidation is connected closely with the prostaglandin-generating system, and it has the potential to modulate the turnover of arachidonic acid and prostaglandin synthesis.     

Letter to the editor: Non steroid anti-inflammatory drugs and pregnancy

A review of relevant clinical research literature leads to the conclusion that any known nonsteroid anti-inflammatory drug (NSAID) should be contraindicated for use during pregnancy. This conclusion is reached as a result of the potentially profound effects of NSAIDs noted on platelet functions and their inhibition of specific tissue prostaglandin(PG) synthetases. Study results have shown that administration of Naproxen to midtrimester abortion patients prolonged the instillation-abortion interval from 33.4 hours +or- 2.9 hours (in a placebo treated group) to 64.7 hours +or- 6.4 hours. Treatment with aspirin prolonged induction-abortion time by about 9 hours and treatment with indomethacin by 30 hours. 2 recent clinical papers have shown that use of acetylsalicylic acid, a relatively weak inhibitor of PG synthetase when compared to Naproxen, by pregnant women at or near term increased their length of gestation, their mean duration of labor, the frequency of postmaturity among them, and the incidence of pre- and postpartum hemorrhage. A small dose of aspirin given to mothers at or near term results in platelet dysfunction in both the mother and the newborn infant. NSAID most frequently disrupts normal hemostatic homeostasis mechanisms. In addition, studies have shown that use of NSAIDs increases the incidence of chromosomal abnormalities in spontaneous abortuses. Such drugs should not be used to prevent threatened abortions.     

Effects of pretreatment with anti-inflammatory drugs on ozone-induced lung damage in rats.

The effects of pretreatment with acetylsalicylic acid (aspirin), hydrocortisone, indomethacin, and heparin administered ip against the pulmonary edema produced by O3-exposure (4 ppm for 4 hr) were studied in rats. These anti-inflammatory drugs were found to alter the injurious effect of O3 on lung differently. First, aspirin at the high dose (125 mg/kg) accentuated O3-induced lung injury, and had no effect at the low dose (10 mg/kg); second, hydrocortisone (50 mg/kg) failed to have any effect; third, indomethacin at a high dose (20 mg/kg) offered a significant degree of protection, but had no effect at the low dose (2.5 mg/kg); and fourth, heparin (1000 units/kg) also offered a significant degree of protection against the lung damage normally induced by O3-exposure. Several mechanisms for the favorable and unfavorable interactions of anti-inflammatory drugs with O3-exposure are discussed.