A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release

Collagen-induced platelet aggregation and thromboxane release is inhibited, in a concentration response relationship, by preincubation of gel-filtered platelets with melatonin in the concentration range 430 nM – 4.3 mM. Inhibition of platelet aggregation and thromboxane release also occurs in the presence of indomethacin (4.3 nM – 4.3 mM), a known potent inhibitor of prostaglandin synthesis. Arachidonic acid-induced platelet aggregation and thromboxane release was inhibited in the presence of 4.0 mM melatonin. We therefore propose that inhibition of prostaglandin synthesis maybe the mechanism by which melatonin expresses its activity. Its antigonadotropic activity may result from inhibition of PGE₂ synthesis in the hypothalamus and median eminence.     

Stimulation of microsomal prostaglandin synthesis by a vasoactive material isolated from blood plasma

Stimulation of prostaglandin synthesis by a material with coronary vasoconstrictor activity extracted from blood plasma was examined. The vasoactive material decreased the Km for arachidonate in the overall synthesis of prostaglandins by rabbit renal microsomal preparations but did not change Vmax. Increases in prostaglandin synthesis caused by the vasoactive material and L-tryptophan or L-epinephrine were additive or synergistic, whereas increases produced by the vasoactive material and hemin or hemoglobin were not. However, hemin and hemoglobin stimulated synthesis of all prostaglandins equally whereas the active material increased the synthesis of prostaglandin F_2α at the expense of other prostaglandins, both in the presence and absence of heme compounds. The increase in prostaglandin F_2α with respect to the other prostaglandins occurred in the presence of reduced glutathione. The vasoactive material attenuated inhibition of prostaglandin synthesis induced by indomethacin or aspirin but not that produced by 5,8,11,14-eicosatetraynoic acid. The interaction of the vasoactive material and indomethacin was competitive whereas hemin attenuated the effects of only low concentrations of indomethacin. Epinephrine enhanced indomethacin inhibition. These data indicate that mode of action of the vasoactive material in prostaglandin synthesis is unlike that of glutathione, aromatic amines, or heme containing compounds.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Bicyclic melatonin receptor agonists containing a ring-junction nitrogen: Synthesis, biological evaluation, and molecular modeling of the putative bioactive conformation

Employing 1,3-dipolar cycloaddition for the synthesis of the 7a-azaindole nucleus, analogues of melatonin have been synthesized and tested against human and amphibian melatonin receptors. Introducing a phenyl substituent in position 2 of the heterocyclic moiety significantly increased binding affinity to both the MT1 and MT2 receptors. Shifting the methoxy group from position 5 to 2 of the 7a-azaindole ring led to a substantial reduction of MT1 binding when MT2 recognition was maintained. We theoretically investigated the hypothesis whether the 2-methoxy function of the azamelatonin analogue 27 is able to mimic the 5-methoxy group of the neurohormone by directing its 2-methoxy function toward the methoxy binding site. DFT calculations and experimental binding differences of analogue compounds indicate that the energy gained by forming the methoxy-specific hydrogen-bond interaction should exceed the energy required for adopting an alternative conformation.     

Indomethacin blocks gonadotropin stimulation of ovarian ornithine decarboxylase activity by inhibiting protein synthesis

Both gonadotropins and prostaglandins stimulate the ornithine decarboxylase (ODC) activity of porcine granulosa cells (1,2). To investigate a possible intermediary role of prostaglandins in this gonadotropin action, the effects of indomethacin on gonadotropin-induced ODC activity were studied. Indomethacin had no effect at concentrations lower than 10⁻⁵ M; at higher concentrations indomethacin exerted a dose-dependent suppression of LH-stimulated ODC activity which was essentially complete at 5 × 10⁻⁴ M. The effects of PGE₂ and 8-Bromo-cAMP, potent stimulators of ODC, were also blocked by indomethacin (5 × 10⁻⁴ M). This effect did not represent direct inhibition of enzyme activity, but appeared to be due to inhibition of protein synthesis by the drug. Thus, incorporation of ¹⁴C-leucine into proteins by these cells was blocked by indomethacin with a dose-response curve similar to that for ODC suppression. This distal effect of indomethacin may complicate the interpretation of some experiments if the inhibitor is assumed to act only at the prostaglandin synthetase step.     

Thrombin,a stimulator of bone resorption

It is shown that thrombin (0.1–7 units/ml) stimulates calcium mobilization and bone matrix degradation, as indicated by release of [³H]proline, from cultured calvarial bones. The second finding in this paper, that indomethacin blocks the stimulating effect of thrombin on bone resorption, is consistent with the concept that prostaglandin synthesis may be involved in this process. It is suggested that thrombin is a potential mediator of bone resorption associated with inflammatory and malignant processes.     

Role of centrally administered melatonin and inhibitors of COX and NOS in LPS-induced hyperthermia and adipsia.

In the present study we have examined the effect of centrally administered non-steroidal anti-inflammatory drugs (NSAIDS), nitric oxide synthase (NOS) inhibitor and melatonin on lipopolysaccharide (LPS)-induced hyperthermia and its anti-dipsogenic effect. Intracerebroventricular (i.c.v.) administration of LPS (100-200 ng/rat) induces a dose dependent elevation in body temperature and decreases water consumption in 24 h water deprived rats. Coadministration of NSAIDS (indomethacin and nimesulide: 10 nM/rat each) with LPS (100 ng) reversed, whereas NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME: 10-20 microg/rat) enhanced LPS-induced hyperthermia. In contrast L-NAME reversed the LPS-induced anti-dipsogenic effect in a dose dependent manner, whereas NSAIDS showed no change in the effect of LPS. Further, centrally administered prostaglandin E2 (PGE2, 0.5-1 microg/rat) produced hyperthermia without affecting the drinking behavior, suggesting that two independent mechanisms operate in LPS-induced hyperthermia and in the anti-dipsogenic effect. The pineal hormone melatonin is known to inhibit cellular damage caused by LPS, produced dose dependent (5-10 nM i.c.v.) inhibition of LPS-induced hyperthermia and adipsia, but failed to reverse the PGE2-induced hyperthermia, shows reversal of LPS-induced hyperthermia by melatonin is due to inhibition of prostaglandin synthesis rather than antagonism of prostaglandin action. The overall study reveals that inhibition of both NO and prostaglandin production by melatonin might be responsible for its reversal of LPS-induced hyperthermia and adipsia.     

The actions of a charged melatonin receptor ligand, TMEPI, and an irreversible MT2 receptor agonist, BMNEP, on mouse hippocampal evoked potentials in vitro

We have previously determined that melatonin modulates hippocampal synaptic transmission in a biphasic way: an initial depression was followed by a recovery/amplification phase. Here we describe the influence of two novel melatonin receptor ligands, BMNEP (N-bromoacetyl-2-iodo-5-methoxytryptamine) and TMPEI (N-[2-(2-Trimethylammoniumethyleneoxy-7-methoxy)ethyl]propionamide iodide), on the population spike (PS) and excitatory postsynaptic potentials (EPSP) recorded from mouse hippocampal slices. BMNEP, which specifically alkylates and constitutively activates the MT2 melatonin receptor, mimicked the first phase of melatonin's action by irreversibly depressing both the PS and EPSP. TMPEI, a charged ligand of plasma membrane melatonin receptors, amplified those potentials in a manner similar to the effect of melatonin observed during the second, recovery phase. Melatonin had no influence on the potentials amplified by the action of TMPEI. Our results suggest that the biphasic, receptor-dependent action of melatonin and its analogs modulates the efficiency of the hippocampal glutamergic synapse and is most likely mediated through two different, sequentially occurring mechanisms.     

Structure-activity studies of melatonin analogues in prepubertal male rats

Comparison has been made between the activity of the pineal hormone melatonin, and several analogues and metabolites in inhibiting sexual development in a protein-restricted prepubertal rat model. Eleven melatonin analogues or metabolites were tested with the aim of evaluating the model as a test of the hypothesis that melatonin acts as a prohormone and that the ring schism metabolites (kynurenamines) mediate many of the effects attributable to melatonin. Although the hypothesis could not be confirmed, modification of the melatonin structure by lengthening the acrylamide side chain or by replacing the 5 methoxy function with fluorine resulted in loss of biological potency. Modification of the melatonin structure to block the two known points of metabolism resulted in no significant alteration in biological activity. Thus 6-chloromelatonin (blocking 6-hydroxylation) and 2,3-dihydromelatonin (blocking oxidative cleavage of the C2-C3 bond) and 6-chloro-2,3-dihydromelatonin remained biologically active. The metabolic products of brain indoleamine-2,3-dioxygenase, N-acetyl-N2-formyl-5-methoxy kynurenamine (aFoMK) and N-acetyl-5-methoxy kynurenamine (aMK), paradoxically were also biologically active.     

Indomethacin inhibits lordosis induced by ring A-reduced progestins: possible role of 3alpha-oxoreduction in progestin-facilitated lordosis

Progestins with a delta-4-3-keto configuration bind to the progestin receptor (PR) and facilitate estrous behavior in estrogen-primed rats. Some ring A-reduced progestins [5alpha-dihydroprogesterone (alphaDHP), allopregnanolone, and epipregnanolone] are more potent estrus-inducing agents than progesterone when iv injected despite their lower affinity for the PR. Yet the estrus-inducing action of such progestins is reduced by the antiprogestin RU486, suggesting that binding to the PR is required for this effect. Because allo- and epi-pregnanolone are oxidized to alpha- and betaDHP, respectively, by 3alpha-hydroxysteroid oxo-reductase (3alphaHSOR), part of their estrus-inducing action may occur through the binding of such DHPs to the PR. Conversely, because 3alphaHSOR reduces alpha- and betaDHP to allo- or epi-pregnanolone, both of which exert membrane effects, the estrus-inducing effect of DHPs may involve actions independent of the PR. To test these possibilities we assessed the effect of indomethacin, a blocker of 3alphaHSOR, on the estrus-inducing action of such progestins. Because indomethacin also inhibits cyclooxygenases, we selected a dose and treatment schedule that does not interfere with prostaglandin-mediated brain processes (e.g., LHRH release). Indomethacin did not significantly modify the effect of progesterone or megestrol acetate on lordosis. Yet, it significantly reduced the action of all ring A-reduced progestins. Results suggest that: (a) oxidation is essential for lordosis facilitation by 3alpha-pregnanolones and (b) reduction of 3-keto progestins generates 3alpha-hydroxy metabolites which synergize with processes triggered by occupation of the PR by 3-keto progestins. The possible participation in this response of other events influenced by indomethacin (e.g., prostaglandin or melatonin synthesis) is discussed.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrenaline, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 μg/ml (0.83 × 10⁻⁵M) for MIX, 18 μg/ml (1 × 10⁻⁴M) for theophylline and 133 μg/ml (6.8 × 10⁻⁴M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyryl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10⁻²M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogeneous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2α on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonism may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.     

Decrease of serotonin N-acetyltransferase activity in rat pineal organs after treatment with prostaglandin synthesis inhibitor indomethacin

Administration of indomethacin to rats abolished the cyclic AMP dependent, dark induced rise in serotonin N-acetyltransferase, presumably by inhibiting prostaglandin synthesis.     

Prostaglandin E(1) inhibits abnormal follicle rupture and restores ovulation in indomethacin-treated rats

In the presence of indomethacin, an inhibitor of prostaglandin (PG) synthesis, the gonadotropin surge induces abnormal follicle rupture at the basolateral follicle sides, thus preventing effective ovulation in rats. This study was undertaken to analyze whether exogenous prostaglandin administration can overcome the antiovulatory action of indomethacin. Cycling rats were treated with vehicle (olive oil) or indomethacin (1 mg/rat) on the morning of proestrus. Rats treated with indomethacin were injected with different doses (50, 250, or 500 micro g/rat) of PGE(1), PGE(2), PGF(2alpha), or vehicle (saline) at 1900 h in proestrus. The ovulatory response was analyzed on the morning of estrus by evaluating follicle rupture and the location of the oocytes in serially sectioned ovaries. The number of oocytes in the oviducts was also counted in rats treated with the highest prostaglandin doses. In indomethacin-treated rats, most newly formed corpora lutea showed abnormal follicle rupture at the basolateral sides. In addition, invasion of the ovarian stroma and blood and lymphatic vessels by granulosa cells and follicular fluid was observed. Prostaglandins of the E series, and especially PGE(1), inhibited abnormal follicle rupture and restored ovulation, although the number of oocytes in the oviducts were significantly decreased. PGF(2alpha) was only partially effective in inhibiting abnormal follicle rupture and restoring ovulation. These data suggest that prostaglandins of the E series, and particularly PGE(1), play a crucial role in ovulation by determining the targeting of follicle rupture at the apex, thus allowing release of oocytes to the periovarian space.     

Stimulation of anterior pituitary prostaglandin E content and somatotropin (growth hormone) synthesis by phospholipase A.

The prostaglandin E content of dispersed rat anterior pituitary glands was found to increase in the presence of phospholipase A or arachidonic acid. The increases were abolished by the addition of indomethacin. Similarly, the rate of somatotropin (growth hormone) synthesis was increased by these two agents, and the increases were again abolished by indomethacin. Phospholipase A also stimulated somatotropin release. The stimulation of prostaglandin E accumulation was a specific response to those fatty acids that are precursors for prostaglandin synthesis. One such precursor, [3H]arachidonic acid, was incorporated by rat anterior pituitary glands in vitro, and found to be associated mainly with phosphatidylethanolamine-like material. It is concluded that the intracellular concentration of prostaglandin E is limited by the availability of precursor fatty acids and that this can be increased by the addition of exogenous precursors or by the action of exogenous phospholipase A on the cellular phospholipid. Factors that increased prostaglandin E concentrations also increase the rate of synthesis of somatotropin, providing further evidence for the concept that prostaglandin E is involved in modulation of the rate of synthesis of this hormone.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. IV. Evidence for participation of prostaglandins of the E series in the early events.

The addition of KLH to KLH-primed rabbit lymph node cell cultures induced an anamnestic antibody response. The further addition of prostaglandins of the E series, but not PGF1^α, enhanced this antibody response manifold. The addition to these cultures of prostaglandin synthetase inhibitors together with KLH inhibited antibody production. At the concentration (10^?4) required to inhibit antibody synthesis, by a variety of criteria one of these inhibitors, indomethacin, was shown not to exert its effects through cytotoxicity. By contrast, two other inhibitors of prostaglandin synthesis, Ro-20-5720 and Ro-3-1314, inhibited antibody synthesis because of their cytotoxicity. The inhibition of the antibody response by indomethacin did not occur when PGE1 or PGE2 was added concurrently to these cultures, clearly showing that inhibition was due to a deficiency of prostaglandins. These findings strongly suggest that induction and/or regulation of the in vitro anamnestic antibody response of KLH-primed lymph node cells to 1 and 100 μg KLH requires continued prostaglandin synthesis. Potential mechanisms for the regulation of the antibody response by prostaglandins are discussed.     

Prostaglandins and myoblast fusion

Physiological concentrations of prostaglandin E₁ (10^?7 and 10^?10M) provoke a discrete burst of cell fusion in cultures of primary chick myoblasts, 5 hr after their addition but well before the start of fusion, under control conditions. Two inhibitors of prostaglandin synthesis, aspirin (2-acetoxybenzoic acid) and indomethacin (1-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid), have been used to examine the possibility of prostaglandin production by the undifferentiated myoblasts. Both inhibitors produce a marked inhibition of cell fusion which is possible to reverse by the further addition of 10^?5M prostaglandin E_↑. The findings provide evidence of prostaglandin synthesis in the cultures and suggest that prostaglandin E_↑ is required for the generation of a transient increase in intracellular cyclic AMP which brings about the cellular changes necessary for fusion to occur.     

Effects of indomethacin on renal inner medullary plasma flow

The effects of the prostaglandin system on renal hemodynamics were studied by treating rats with a single intraperitoneal dose of indomethacin, an inhibitor of prostaglandin synthesis. Medullary plasma flow was significantly reduced 30–45 minutes after indomethacin, but was elevated 3–6 hours after indomethacin. These changes in medullary plasma flow correlated well with circulating levels of prostaglandins A and E. Total renal blood flow decreased following indomethacin treatment, but returned to normal levels within an hour. These results indicate that the inhibition of prostaglandin synthesis following a single intraperitoneal dose of indomethacin is short-lived and is followed by a significant elevation in prostaglandin synthesis. It is likely that prostaglandin levels play an important role in the control of renal medullary plasma flow.     

Differential effects of ibuprofen,indomethacin, and meclofenamate on prostaglandin endoperoxide H2 metabolism

Summary Previous studies have suggested the possibility that the non-steroidal antiflammatory drug (NSAID), ibuprofen, may inhibit thromboxane (TX) A₂ synthase activity in addition to inhibiting cyclooxygenae activity. Microsomal fractions isolated from the cat lung contain cyclooxygenase as well as prostacyclin (PGI₂) synthase, TX synthase, and a GSH-dependent prostaglandin (PG) E₂ isomerase activities. When [1-¹⁴C] PG endoperoxide H₂ (PGH₂) was used as substrate, ibuprofen, indomethacin, and meclofenamate exhibited differential effects on terminal enzyme activities. Ibuprofen, at concentrations up to 1 mM, had no effect on the activities of PGI₂ synthase, TXA₂ synthase of GSH-dependent PGE₂ isomerase, whereas indomethacin selectively inhibited PGI₂ synthase activity at 5 x 10^–4 M and 10^–3 M. Meclofenamate selectively inhibited TXA₂ synthase activity at 5 x 10^–4 M and 10^–3 M. At concentrations of 5 x 10^–3 M, this selectivity was not oberved, and indomethacin and meclofenamate decreased the formation of both 6-keto-PGF₁ and TXB₂. These data indicate that the choice of NSAID and the concentration employed may specifically alter PGH₂ metabolism. This action may affect the physiologic consequences of the exchange of PGH₂ between cells. The data further indicate that indomethacin has the potential for use as a tool to specifically attenuate PGI₂ synthase activity in vitro.     

Effect of sparteine sulfate on uterine prostaglandin F in the rat

Indomethacin, an inhibitor of prostaglandin synthetase, added to an bath in a concentration of 1, 5, and 10 × 10⁻⁶ g/ml reduced sparteine-induced contractions of isolated uterine segments from pregnant rats. Contractions induced by prostaglandin F₂α and acetylcholine were not reduced.Sparteine increased the prostaglandin F content of the blood and uterine tissue in the pregnant but not in the nonpregnant rat. This increase was significantly reduced by the administration of indomethacin (10 mg/kg). The present study suggests that the mechanism of sparteine action is mediated through a prostaglandin F system.     

A biochemical hypothesis to explain the mechanism of luteal regression

It seems likely that luteal regression may involve a direct biochemical action of prostaglandin F₂α (PGF₂α) on the luteal cell since there are now several reports that PGF₂α can directly inhibit steroidogenesis . However, the mechanism of such an action of PGF₂α remains obscure.This article initially reviews the central role of adenosine 3,^I5^I-mono-phosphate (c-AMP) in initiating and maintaining the structural and functional changes occurring on luteinisation. A mechanism is suggested, supported by results obtained using granulosa cells in tissue culture, in which PGF₂α initiates functional luteolysis by inhibiting further synthesis of c-AMP. This mechanism is then used in conjunction with further observations to provide a possible explanation for the inability of PGF₂α to regress newly formed corpora lutea. Finally, the possible mechanisms of structural regression are discussed.