Biomedical subjects

P Delagrange

Publications and source records attributed to P Delagrange.

At least 19 recordsLinked to original sources

PubMed2000

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

Amino Acid Sequence↗

PubMed2000

Anxiolytic-like properties of melatonin receptor agonists in mice: involvement of mt1 and/or MT2 receptors in the regulation of emotional responsiveness.

The anxiolytic-like properties of melatonin have been established in rodents. The present study investigated the possible involvement of melatonin receptors/binding sites in the regulation of emotional responsiveness in mice, using an mt1/MT2 receptor specific agonist (S 23478) and two specific ligands of MT3 binding sites with agonistic properties (N-acetylserotonin (NAS) and 5-methoxycarbonylamino N-acetyltryptamine (5-MCA-NAT)). We examined the behavioural effects of these compounds in C3H/He mice confronted with two anxiety models: the free-exploratory test, in which C3H/He mice present neophobic reactions ("trait" anxiety), and the light/dark choice test, which is an unconditioned conflict test (inducing "state" anxiety). Melatonin and S 23478 decreased anxious reactions in both the free-exploratory test (5-25 mg/kg) and the light/dark choice test (melatonin: 20 mg/kg; S 23478: 10-20-40 mg/kg). NAS exerted anxiolytic-like effects only at a dose of 35 mg/kg in the free-exploratory test and at a dose of 40 mg/kg in the light/dark choice test. Finally, 5-MCA-NAT was devoid of anxiolytic-like effects in both tests. These results suggest that the anxiolytic properties of melatonin could involve the activation of mt1 and/or MT2 receptors rather than of the MT3 binding site.

Analysis of Variance↗

PubMed2000

Chimeric melatonin mt1 and melatonin-related receptors. Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt1 receptor.

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

Animals↗

PubMed2000

Substrate specificity and inhibition studies of human serotonin N-acetyltransferase.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.

Acyl Coenzyme A↗

PubMed2000

Characterisation of human melatonin mt(1) and MT(2) receptors by CRE-luciferase reporter assay.

A cyclic AMP response element (CRE)-luciferase reporter gene assay was used to characterise the functional responses of human melatonin mt(1) and human melatonin MT(2) receptors, stably expressed in the human embryonic kidney cell line HEK293, to a series of six naphthalenic analogues of melatonin. By comparison to the observed melatonin-mediated inhibition of stimulated luciferase levels the naphthalenic series was identified as comprising agonists, partial agonists and one antagonist of melatonin mt(1) and melatonin MT(2) receptor function. Three of the agonist/partial agonist members of this series were also identified as displaying a functional selectivity for the melatonin MT(2) receptor. Competitive displacement of 2-[125I]iodomelatonin binding to the ovine pars tuberalis melatonin ML(1) receptor demonstrated a close correlation to the observed functional luciferase responses of the human melatonin mt(1) receptor. We conclude that the CRE-luciferase reporter gene assay provides an effective functional screening method for the pharmacological characterisation of human melatonin receptor subtypes.

Acetamides↗

PubMed2000

Pharmacological studies on the inhibitory action of melatonin and putative melatonin analogues on porcine vascular smooth muscle.

The effect of high concentrations of melatonin, and related indole-based and naphthalene-based derivatives, has been examined in the porcine coronary artery, pulmonary artery and the marginal artery of the colon. In addition, we have pharmacologically examined the role of cyclic GMP in the relaxatory action of these agents. Cumulative addition of melatonin (3-300 microM) caused a slowly developing relaxation in all three vascular preparations pre-contracted with 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2alpha (U46619), a thromboxane mimetic agent. The estimated pIC50 values were 4.10-3.70 (coronary artery), 3.89 (pulmonary artery) and 3.96 (marginal artery). All melatonin analogues examined also produced concentration-dependent inhibition of U46619-induced contractions of the coronary and marginal arteries in a qualitatively similar manner to melatonin. The rank order of potency (based on the pIC50 values) of these compounds in both vascular tissues was N-[2-(3-ethyl-7-methoxynaphthyl) ethyl]-acetamide (S21634) >2-iodomelatonin = N-[2-(7-methoxynaphth-1-yl)-ethyl]-acetamide (S20098) = N-[2-naphth-1-yl-ethyl]-cyclobutyl carboxamide (S20928) >melatonin >N-acetyl-5-HT. Finally, the pharmacological characteristics of melatonin and S21634 as phosphodiesterase inhibitors were compared to those of zaprinast, a known cyclic GMP-specific phosphodiesterase inhibitor. Zaprinast also caused concentration-dependent inhibition of U46619-induced tone. All three compounds, zaprinast (10 microM), melatonin (300 microM) and S21634 (30 microM), significantly enhanced sodium nitroprusside-induced relaxations. The inhibitory action of zaprinast per se was greater in the presence of the endothelium and significantly attenuated by 3 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a selective inhibitor of guanylyl cyclase. In marked contrast, the vasorelaxant action of melatonin and S21634 was not affected by the removal of the endothelium or the addition of ODQ. In summary, we have shown that porcine arterial smooth muscle relaxes in response to high concentrations of melatonin and other related melatonin receptor ligands. However, it appears that the receptive site is pharmacologically different from the melatonin receptors currently known and does not involve inhibition of cyclic GMP-specific phosphodiesterase.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Substituted oxygenated heterocycles and thio-analogues: synthesis and biological evaluation as melatonin ligands.

A new series of substituted oxygenated heterocycles and thio-analogues were synthesized and evaluated as melatonin receptor ligands. The replacement of the indolic moiety of melatonin by heterocyclic skeleton such as 1,4-benzodioxin, 2,3-dihydro-1,4-benzodioxin, chroman, 2,3-dihydro-1,4-benzoxathiin, thiochroman, carrying the amidic chain on the aromatic ring, leads to compounds showing a weak affinity for melatonin receptors, except for the compounds 1cb and 1hb.

Animals↗

PubMed2000

Synthesis of a small library of phenylalkylamide derivatives as melatoninergic ligands for human mt1 and MT2 receptors.

Focused small libraries of melatonin receptor ligands from arylalkylamine derivatives were synthesised by combinatorial chemistry using the mix and split method in the solid phase. A library of 108 compounds was then synthesised from 12 arylalkyl amines and nine carboxylic acids. The compound mixtures were evaluated on chicken brain melatonin and recombinant human mt1 and MT2 receptors. Deconvolution of the most potent mixture demonstrated the superiority of 3-methoxy and 2,5-dimethoxy substitution on the phenyl ring with isopropyl, propyl and ethyl amido chains. Several compounds with nanomolar affinity for human melatonin receptors were obtained.

Amides↗

PubMed2000

Effect of melatonin on motility pattern of small intestine in rats and its inhibition by melatonin receptor antagonist S 22153.

Melatonin is synthesized during the night by the pineal gland. Recently, melatonin binding sites have been identified in the gut. Despite few studies, the physiological role of melatonin in gut function remains unclear. The objective of the present study was to investigate the effects of melatonin in the regulation of intestinal motility by using the melatonin receptor antagonist S 22153 in rats. Twenty-four male Wistar rats (400 +/- 25 g) were equipped with intraparietal electrodes along the small intestine. Rats were subjected to a 12:12 hr light:dark schedule. During the dark phase, intestinal migrating motor complexes (MMCs) frequency increased (P < 0.05) by 20% in the duodenum and in the jejunum compared with daylight. This effect is due to a significant reduction in the irregular spiking activity (ISA) of MMCs. Concurrently, at night, the duration of the postprandial motor response is reduced by 30% in the duodenum and 50% in the jejunum and ileum. The administration of S 22153 (2 mg/kg sc) at night suppressed these nocturnal variations and restored the daylight values. In contrast, S 22153 was ineffective during daylight whatever the digestive state. Administration of melatonin (1 mg/kg iv) during the preprandial state, 3 hr after light onset, decreased (-80%) the duration of the ISA of MMCs at the three intestinal levels. During the satiety phase, melatonin administered 10 min before or 15 min after food onset induced the appearance of a transitory preprandial-like motor profile in the entire small intestine. In contrast, when administered at the end of the meal it was ineffective. Preprandial and postprandial melatonin effects were prevented by S 22153 pretreatment. In conclusion, these findings reveal, first, that endogenous melatonin is physiologically involved in the pre- and postprandial changes of intestinal motility at night. Second, exogenous melatonin produces pharmacological effects on pre- and postprandial intestinal motility. In both cases, the action of melatonin corresponds to an inhibition of ISA and a reinforcement of the cyclic MMC pattern.

Action Potentials↗

PubMed2000

Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice.

Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects.

Animals↗

PubMed2000

Screening of ligand binding on melatonin receptor using non-peptide combinatorial libraries.

The screening of combinatorial libraries requires a deconvolution procedure to obtain, in fine, the most active compound of the starting library. The standard screening assays used in regular molecular pharmacology, have been poorly assessed when transposed to combinatorial chemistry-related experiments, particularly those involving large numbers of chemicals in a single assay. One key issue is the effect of the inactive analogs on the identification of the active ligand in mixtures. We chose melatonin receptors to measure the apparent affinity of a single ligand when tested alone or in mixtures of non-peptide low molecular weight compounds. Using ligands with IC50 from the micro- to the picomolar range, mixed with increasingly complex mixtures of 5 to 20 or 25 inactive compounds, we analyzed the displacements from the mt1 and MT2 melatonin receptor subtypes of the radioligand 2-iodomelatonin (KD= 25 pmol/l and 200 pmol/l, respectively) . The behavior of equimolar mixtures in displacement curves led to the conclusion that the observed binding affinity reflects the dilution effect of mixing the active component with inactive compounds but does not reveal noticeable interactions which would interfere with the binding process. From the practical point of view, the concentrations of the active species in the binding assay should be large enough to displace significantly the radioligand, a requirement which may be limited by the solubility of the ligand mixtures. In contrast, previous observations with peptide libraries report that the dilution effect is often compensated by additive or synergic action of structurally related analogs, thus making possible the deconvolution of very large (typically up to 10(7) compounds) peptide libraries.

Binding, Competitive↗

PubMed1999

A single oral dose of S 22153, a melatonin antagonist, blocks the phase advancing effects of melatonin in C3H mice.

Disorders of the circadian system have been associated with adverse mental and physical conditions, raising the possibility that pharmacological agents acting on the circadian system could have therapeutic benefit. Compounds acting as agonists or antagonists of melatonin, an endogenous hormone able to feed back on the circadian clock, are currently under development for possible use in modulating circadian rhythmicity. In the present study, we examined the ability of an oral dose of S 22153, a synthetic melatonin antagonist, to block the phase advancing effect of a melatonin injection at circadian time 10 in free running C3H mice. Our results show that S 22153 had no effect per se on the phase or the period of the locomotor activity rhythm but was able to block the phase advancing effect of melatonin, suggesting potent antagonist effects at melatonin receptors. Availability of a melatonin antagonist may yield new insight into the role of melatonin in physiological processes and such compounds may find widespread clinical applications.

Administration, Oral↗

PubMed1999

Melatonergic properties of the (+)- and (-)-enantiomers of N-(4-methoxy-2,3-dihydro-1H-phenalen-2-yl)amide derivatives.

N-(4-Methoxy-2,3-dihydro-1H-phenalen-2-yl)amide derivatives, conformationally restricted ligands for melatonin receptors, were synthesized by an alternative synthetic method from the corresponding 1,8-naphthalic anhydride which was transformed into the phenalenecarboxylic acid 7. A Curtius reaction on 7 gave the amino compound which was acylated to give compounds 4a-c. The (+)- and (-)-4a-c enantiomers were separated by semipreparative chiral HPLC. Compounds were evaluated for their affinity for chicken brain melatonin receptors in binding assays using 2-[125I]iodomelatonin and for their potency to lighten the skin of Xenopus laevis tadpoles. The butyramido derivative 4c was the most potent ligand (Ki = 1.7 nM). No enantioselectivity was observed with the enantiomers which were equipotent to the racemic mixture. In contrast to the reference compounds, melatonin, agomelatine (S 20098), and N-[2-(2, 7-dimethoxynaphth-1-yl)ethyl]acetamide, which were very potent at lightening the skin of X. laevis tadpoles, compounds 4a-c were inactive or weakly active (EC50 > 1 microM). In this bioassay, compound 4a was characterized as a putative antagonist of melatonin receptors.

Acetamides↗

PubMed1999

Effects of melatonin on neophobic responses in different strains of mice.

Anxiolytic properties of melatonin in rodents had usually been examined in behavioral tests based on stressful situations, i.e., in animal models of "state" anxiety. However, no study reports effects of melatonin on emotionality of rodents submitted to situations devoid of stressful components as in the free-exploratory test, which gives to animals the opportunity to choose freely between familiar and unfamiliar places. This procedure has been proposed as a method for measuring an endogenous form of anxiety called "trait" anxiety. The present study first investigated the effects of melatonin on neophobic responses of male C57BL/6, C3H/He, and BALB/c mice submitted to a free-exploratory test. Results demonstrated that melatonin had no effect in C57BL/6 mice that presented very low neophobic responses, whereas it was effective in reducing neophobia of BALB/c and C3H/He mice that presented, respectively, strong and intermediate avoidance responses towards unfamiliarity. Indeed, mice of both latter strains treated with melatonin made fewer attempts to enter into the unfamiliar compartment, exhibited a lower latency of the first entry into the unfamiliar places, and spent more time in them. Thus, melatonin appeared to be equally effective in reducing "trait" anxiety in both BALB/c and C3H/He mice. Moreover, flumazenil was able to counteract, in a dose-dependent manner, the anxiolytic activity of melatonin in BALB/c, suggesting involvement of central GABAergic system in the pharmacological effects of melatonin.

Animals↗

PubMed1999

Antagonistic effects of S 22153, a new mt1 and MT2 receptor ligand, on the neophobia-reducing properties of melatonin in BALB/c mice.

When exposed to a free-exploratory situation, BALB/c mice are well known to exhibit strong avoidance responses toward unfamiliar places (neophobia). Because melatonin was found to significantly reduce neophobia in BALB/c mice, it seemed interesting to examine potential antagonistic effects of S 22153, a new melatonin mt1 and MT2 receptor ligand, on the neophobia-reducing properties of melatonin in BALB/c mice confronted with the free-exploratory paradigm. S 22153 was able to block, in a dose-dependent manner, the anxiolytic-like properties of melatonin when it was administered 5 min before melatonin. The antagonistic effects of S 22153 persisted when the drug was administered 2 or 4 h before melatonin, and were almost abolished when it was administered 6 h before melatonin. These results suggest that the anxiolytic-like effects of melatonin on the neophobic responses in BALB/c mice are mediated by mt1 and/or MT2 receptors.

Animals↗

PubMed1999

Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats.

1. In this study reverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4-phenyl-2-propionamidotetraline (4-P-PDOT) and 2-benzyl-N-pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. 2. Using RT-PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. 3. At low concentrations of the MT2 selective ligands, neither 10 nM 4-P-PDOT (pEC50=8.70+/-0.31 (control) vs 8.73+/-0.16, n=6) nor 60 nM 2-benzyl-NV-pentanoyltryptamine (pEC50= 8.53+/-0.20 (control) vs 8.83+/-0.38, n = 6) significantly altered the potency of melatonin in the rat tail artery. 4. At concentrations non-selective for mt1 and MT2 receptors. 4-P-PDOT (3 microM) and 2-benzyl-N-pentanoyltryptamine (5 microM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4-P-PDOT, the estimated pKB (6.17+/-0.16, n=8) is similar to the binding affinity for mt1 receptor. 5. Pre-incubation with 1 microM melatonin did not affect the conversion of [3H]-adenine to [3H]-cyclic AMP under basal condition (0.95+/-0.19% conversion (control) vs 0.92+/-0.19%, n=4) or following exposure to 30 microM forskolin (5.20+/-1.30% conversion (control) vs 5.35+/-0.90%, n=4). 6. Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

Animals↗

PubMed1999

Effects of melatonin on rat pial arteriolar diameter in vivo.

1. Based on our finding that melatonin decreased the lower limit of cerebral blood flow autoregulation in rat, we previously suggested that melatonin constricts cerebral arterioles. The goal of this study was to demonstrate this vasoconstrictor action and investigate the mechanisms involved. 2. The effects of cumulative doses of melatonin (10-10 to 10-6 M) were examined in cerebral arterioles (30 - 50 microM) of male Wistar rats using an open skull preparation. Cerebral arterioles were exposed to two doses of melatonin (3x10-9 and 3x10-8 M) in the absence and presence of the mt1 and/or MT2 receptor antagonist, luzindole (2x10-6 M) and the Ca2+-activated K+ (BKCa) channel blocker, tetraethylammonium (TEA+, 10(-4) M). The effect of L-nitro arginine methyl ester (L-NAME, 10-8 M) was examined on arterioles after TEA+ superfusion. Cerebral arterioles were also exposed to the BKCa activator, NS1619 (10(-5) M), and to sodium nitroprusside (SNP, 10-8 M) in the absence and presence of melatonin (3x10-8 M). 3. Melatonin induced a dose-dependent constriction with an EC50 of 3.0+/-0.1 nM and a maximal constriction of -15+/(-1%). Luzindole abolished melatonin-induced vasoconstriction. TEA+ induced significant vasoconstriction (-10+/(-2%). No additional vasoconstriction was observed when melatonin was added to the aCSF in presence of TEA+, whereas L-NAME still induced vasoconstriction (-10+/(-1%). NS1619 induced vasodilatation (+11+/(-1%) which was 50% less in presence of melatonin. Vasodilatation induced by SNP (+12+/(-2%) was not diminished by melatonin. 4. Melatonin directly constricts small diameter cerebral arterioles in rats. This vasoconstrictor effect is mediated by inhibition of BKCa channels following activation of mt1 and/or MT2 receptors.

Algorithms↗

PubMed1999

Microdialysis of melatonin in the confluens sinuum of the rat following quantification by gas chromatography-mass spectrometry in the negative chemical ion mode.

In this study, an original surgical implantation technique in the confluens sinuum via the superior sagittal vein was developed to quantify melatonin secretion by the pineal gland. Melatonin (CAS 73-31-4) was determined using gas chromatography couples to negative ion chemical ionisation mass spectrometry following liquid extraction and derivatisation by penta-fluoropropionic acid anhydride (PFPA). The minimum detectable amount was 40 fg per injection, corresponding to 1 pg.ml-1 in dialysate. The assay was linear in the range 20-1000 pg.ml-1. This method was suitable for routine melatonin determination in dialysats of peripheral and central circulation with coefficients of variation of 11.2 and 24.6%, respectively for within and between analyses. Profiles of melatonin concentration were obtained (n = 3 rats) over a 2-day experimentation with a slowly diminution of the filtration capacity of the probe during the second day. The nocturnal concentrations of melatonin in the confluens sinuum dialysat ranged from 1003.9 to 2345 pg.ml-1 in the dialysat, indicating wide interindividual variations in the melatonin levels.

Animals↗