Biomedical subjects

D Sugden

Publications and source records attributed to D Sugden.

At least 19 recordsLinked to original sources

PubMed1995

Mapping the melatonin receptor. 3. Design and synthesis of melatonin agonists and antagonists derived from 2-phenyltryptamines.

Three series of 2-phenyltryptamides were prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor in chicken brain and in Xenopus laevis melanophore cells. The 5-methoxy-2-phenyltryptamides (6a-j) have high binding affinities for the chicken brain receptor, in some cases (6a-d) greater than that for melatonin, confirming and extending the work of Spadoni et al., and act as agonists in the Xenopus melanophore assay. Analogues lacking the 5-methoxyl group (2a-n) had a considerably lower affinity for the chicken brain receptor. In the Xenopus melanophore assay the compounds acylated on nitrogen by an alkyl group (2a-d) were agonists whereas the compounds acylated on nitrogen by an alicyclic group (2f-i) were antagonists. Introducing a methyl group at N1 (7) led to an increase in binding affinity in the chicken brain assay, whereas introducing an ethyl group (13) led to a decrease in binding affinity. A methyl substituent at the beta-position of the 3-amidoethane side chain (8, 11) also led to an increase in the binding affinity. The only analogue acylated on nitrogen with an alkyl group (acetyl) which showed antagonist activity was 9, which has a beta-methoxymethyl side chain. In the absence of the 5-methoxyl group the methoxymethyl function may cause the molecule to bind in a different configuration so that it is no longer able to activate the receptor. All of these observations are in agreement with a model of melatonin at the receptor site in which the 3-amidoethane side chain is in a conformation close to the 5-methoxyl group.

Animals↗

PubMed1995

Sedative potency and 2-[125I]iodomelatonin binding affinity of melatonin analogues.

Melatonin (5-methoxy N-acetyltryptamine), the hormone synthesized and released from the pineal gland each night, has sedative and sleep-promoting effects in experimental animals and man. In the present study, the sedative effect of melatonin and a number of analogues was determined by examining their ability to extend the duration of the loss of righting reflex ("sleeping time") in mice injected with pentobarbitone (50 mg/kg i.v.). All of the analogues tested produced a dose-related (5-20 mg/kg) potentiation of pentobarbitone sleeping time. In radioligand binding assays using 2-[125I]iodomelatonin in chicken brain membranes, all of the analogues were competitive inhibitors. There was no correlation between their ability to inhibit 2-[125I]iodomelatonin binding in chick and sedative potency in the mouse. Potentiation of pentobarbitone sleeping time by diazepam (1 mg/kg i.p.), but not melatonin (10 mg/kg i.p.), was blocked by pretreatment with the benzodiazepine antagonist, flumazenil (10 mg/kg i.p.). Similarly, an increase in pentobarbitone sleeping time produced by the aminoalkylindole cannabinoid receptor agonist, WIN 55212-2 (0.5 mg/kg i.p.), but not that produced by melatonin (10 mg/kg i.p.) was reduced by the cannabinoid receptor antagonist WIN 56098 (5 mg/kg i.p.). These studies confirm that melatonin has sedative activity and show that this action is shared by several structurally-related analogues but does not appear to be mediated by an interaction with benzodiazepine or cannabinoid receptors.

Animals↗

PubMed1995

Interlimb coupling in children with hemiplegic cerebral palsy.

Children with hemiplegic cerebral palsy participated in three experiments designed to examine interlimb coupling during reaching and pointing/grasping tasks. Comparisons were made between the hands working in isolation and working together. Descriptions of the movements involved both qualitative and quantitative methods, using normal video analysis and kinematic analysis. In order to obtain a total picture of reaching and grasping it was necessary to examine multiple variables both qualitatively and quantitatively. There was strong evidence of coupling between the hands, with coupling of timing being the most prevalent followed by coupling of trajectory and hand posture. All children showed some form of coupling in the experiments.

Adolescent↗

PubMed1995

Structural requirements at the melatonin receptor.

1. High affinity, specific binding sites for the pineal hormone, melatonin (5-methoxy N-acetyltryptamine) can be detected in chick brain membranes by use of the radiolabelled agonist, 2-[125I]-iodomelatonin (2-[125I]-aMT). 2. The affinity of a number of analogues of melatonin at the 2-[125I]-aMT binding site was determined and compared with an analysis of their electronic structure and significant quantitative relationships obtained. 3. The best correlations indicated that binding affinity was correlated with delta E, the difference between the frontier orbital energies, and QNH, the electron density in the highest occupied molecular orbital of the side-chain nitrogen atom. 4. These findings suggest that ligand binding may involve hydrogen bonding between the 5-methoxy and amide moieties of melatonin and complementary amino acid residues, and charge transfer interactions between the indole ring of melatonin and an aromatic amino acid in the receptor binding site. 5. A molecular model of a putative binding site is proposed based on the predicted amino acid sequence of the cloned Xenopus laevis melanophore melatonin receptor and the quantitative structure-affinity relationships observed in the present study.

Animals↗

PubMed1994

N-acyl-3-amino-5-methoxychromans: a new series of non-indolic melatonin analogues.

A novel series of melatonin analogues is described which are based on the chroman nucleus. These N-acyl-3-amino-5-methoxychromans competitively inhibit [125I]2-iodomelatonin binding to chicken brain membranes although with reduced affinity compared to melatonin. The slope of the competition curves suggests the interaction of the chromans with a single binding site. On cultured Xenopus laevis melanophores, the chroman analogues produce different responses; N-chloroacetyl-3-amino-5-methoxychroman (ClaMCh), like melatonin, is a full agonist at the melanophore receptor and produces a complete aggregation of pigment granules. In contrast, N-acetyl- and N-cyclopropyl-3-amino-5-methoxychroman have no agonist activity, while N-propionyl- and N-butanoyl-3-amino-5-methoxychroman produce only partial aggregation of pigment. ClaMCh is 40-fold weaker at inducing pigment aggregation in melanophores (EC50 = 15 microM) than in inhibiting [125I]2-iodomelatonin binding in chicken brain membranes (Ki = 0.38 microM) suggesting that this analogue may discriminate between melanophore and chicken brain melatonin receptors. Chroman-based melatonin analogues may be useful tools for characterizing potential melatonin receptor subtypes.

Animals↗

PubMed1994

Thermodynamic analysis of agonist and antagonist binding to the chicken brain melatonin receptor.

1. The binding of 2-[125I]-iodomelatonin to chicken brain membranes, and the inhibition of binding by melatonin, N-acetyltryptamine and luzindole, were examined at temperatures between 4 degrees C and 37 degrees C. 2. At all temperatures studied, the binding affinity (Kd or Ki) for 2-[125I]-iodomelatonin, melatonin (both agonists) and, to a lesser extent, N-acetyltryptamine (a partial agonist) was reduced by inclusion of guanosine triphosphate (GTP, 1 mM) in the assay. GTP did not affect the Ki for luzindole, a melatonin receptor antagonist. 3. The maximal density of binding sites (Bmax) was not affected by temperature but the Kd showed a peak at 21 degrees C with lower values at both higher and lower temperatures giving curvilinear van't Hoff plots (lnKA vs l/temperature). 4. Derived changes in entropy (delta S degree) and enthalpy (delta H degree) of binding for all of the melatonin ligands decreased as temperature increased. 5. The affinity, and thus the free energy of binding, delta G degree, of these ligands at the melatonin receptor have identical values at several temperatures yet at these temperatures delta S degree and delta H degree were very different, implying that more than one intermolecular force must be involved in the binding of ligand and receptor. 6. Conceivably, the large positive delta S degree observed at low temperatures, perhaps as a result of hydrophobic interactions, is compensated by a corresponding, but opposite, change in enthalpy at higher temperatures. However, it is not clear what type of binding force(s) would show such a temperature-dependence. 7. These studies suggest that caution must be exercised in the molecular interpretation of derived measures of delta S degree and delta H degree obtained from direct measurements of delta G degree.

Animals↗

PubMed1992

The ontogeny of 2-[125I]iodomelatonin binding sites in chicken brain.

The characteristics of the binding sites for 2-[125I]iodomelatonin were studied in chicken brain membranes during development. Specific binding, defined using cold melatonin (1 microM), was detected as early as 8-day-old embryos. Scatchard analysis of saturation experiments showed that 2-[125I]iodomelatonin binds to a single class of site at all ages tested (8-day-old embryos to 3-month-old chicks). Binding affinity (Kd) did not change during development (18-31 pM), but the maximal number of binding sites (Bmax) increased until embryonic day 18, and then remained relatively constant until 30 days of age. A further increase in Bmax was seen at 3 months of age. Guanosine 5'-triphosphate (GTP, 1 mM) inhibited 2-[125I]iodomelatonin binding at all ages suggesting that the melatonin binding site is coupled to a guanine nucleotide binding protein at a very early stage of development. Competition experiments with a number of melatonin analogues indicated that the binding site detected in the brain at embryonic day 8 was pharmacologically identical to that observed 15 days after hatching.

Aging↗

PubMed1992

Effect of putative melatonin receptor antagonists on melatonin-induced pigment aggregation in isolated Xenopus laevis melanophores.

The ability of putative melatonin receptor antagonists to antagonise melatonin-induced aggregation of pigment granules in cultured neural crest Xenopus laevis melanophores was examined. Neither ML 23 (N-(2,4-dinitrophenyl)-5-methoxytryptamine) nor 6-methoxy-2-benzoxazolinone showed agonist or antagonist activity. N-Acetyltryptamine and N-butanoyltryptamine were partial agonists; both compounds aggregated pigment granules in some cells, but also reversed melatonin-induced pigment agreggation in a fraction of the cells tested. In contrast, 2-benzyl N-acetyltryptamine (luzindole) did not show agonist activity (upto 10 microM) but did reverse the aggregating action of melatonin at 1 and 10 microM. Pretreatment of melanophores with luzindole shifted the melatonin concentration-response curve to the right.

Animals↗

PubMed1992

Characterization of 2-[125I]iodomelatonin binding sites in the brain of a marsupial, Bennett's wallaby (Macropus rufogriseus rufogriseus).

1. Specific high affinity binding of 2-[125I]iodomelatonin was detected in the brain of the pouch young of a marsupial, Bennett's wallaby. 2. Binding was rapid, stable, saturable and reversible. 3. Scatchard analysis indicated a single class of high affinity binding sites with an equilibrium dissociation constant (Kd) of 68 +/- 13 pM, a maximal number of binding sites (Bmax) of 0.7 +/- 0.1 fmol/mg protein and a Hill coefficient (nH) of 1.12 +/- 0.10. 4. Specific binding was inhibited by GTP (1 mM) indicating that the melatonin receptor is coupled to a guanine nucleotide binding protein, and by melatonin and closely related analogues with a potency order identical to that reported previously in the brain of eutherian mammals, birds and a reptile. 5. These studies suggest that the melatonin receptor is well-conserved through evolution.

Animals↗

PubMed1992

Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores.

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.

Adenosine Monophosphate↗

PubMed1991

Pharmacological identity of 2-[125I]iodomelatonin binding sites in chicken brain and sheep pars tuberalis.

2-[125I]Iodomelatonin was used to compare the binding and pharmacological characteristics of the melatonin receptor sites found in chicken brain and sheep pars tuberalis. Scatchard analysis and kinetic experiments showed that 2-[125I]iodomelatonin binds to a single class of site in both tissues with high affinity (Kd 20-34 pM). Competition experiments, using 21 analogues of melatonin, gave inhibition constants (Ki) for the two sites which were significantly correlated (r = 0.985, n = 21, P less than 0.0001). We conclude that the 2-[125I]iodomelatonin binding sites in sheep pars tuberalis and chicken brain have identical binding and pharmacological characteristics.

Animals↗

PubMed1991

Down-regulation of pinealocyte protein kinase C: effect on alpha 1-adrenergic potentiation of beta-adrenoceptor stimulation of cyclic AMP accumulation and induction of serotonin N-acetyltransferase activity.

Treatment of rat pinealocytes with 4 beta-phorbol 12,13-dibutyrate down-regulated protein kinase C (PKC) activity. Loss of activity was concentration-dependent (50% loss at 8 x 10(-7) M after 18 h of treatment) and time-dependent (50% loss after 2 h with 3 x 10(-6) M). Phenylephrine, an alpha 1-adrenergic agonist, and phorbol esters unable to activate PKC did not down-regulate the enzyme. alpha 1-Adrenergic amplification of beta-adrenergic stimulation of cyclic AMP accumulation, a response previously shown to be mediated by PKC activation, was reduced by only 50% in cells in which PKC activity was down-regulated by approximately 95%. These data suggest that there is not a simple proportional relationship between the degree of activation of pinealocyte PKC and the alpha 1-adrenergic amplification of beta-adrenergic cyclic AMP synthesis. In down-regulated cells, alpha 1-adrenergic amplification of beta-adrenergic induction of serotonin N-acetyltransferase activity, a key cyclic AMP-responsive enzyme involved in the nocturnal synthesis of the pineal hormone melatonin, was unchanged. Thus, even though alpha 1-adrenergic amplification of cyclic AMP synthesis is impaired, sufficient cyclic AMP is generated to allow a full induction of serotonin N-acetyltransferase activity. This finding raises the important question of whether the alpha 1-adrenergic amplification mechanism has a physiological role in regulating melatonin synthesis in vivo.

Adrenergic alpha-Agonists↗

PubMed1991

Guanine nucleotides regulate 2-[125I]iodomelatonin binding sites in chick retinal pigment epithelium but not in neuronal retina.

The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 +/- 55 pM and a total number of binding sites (Bmax) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the KD was 34.1 +/- 2.2 pM and the Bmax 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin greater than 2-chloromelatonin greater than melatonin greater than 6-chloromelatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than 6-methoxyharmalan greater than 5-hydroxytryptamine. Linear regression of log Ki values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p less than 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.

Adenine Nucleotides↗

PubMed1991

Aggregation of pigment granules in single cultured Xenopus laevis melanophores by melatonin analogues.

1. Isolated melanophores were differentiated from aggregates of neural crest obtained from neurula stage Xenopus laevis embryos after 2 days in culture. 2. Condensation of pigment granules in these cells by melatonin (5-methoxy N-acetyltryptamine, aMT) and various novel analogues was monitored with an image analysis system to quantitate the area occupied by pigment in individual cells. 3. Melanophores exposed to vehicle (a maximum of 0.1% MeOH) showed little (less than 5%) change in pigment area. aMT produced a dramatic condensation of pigment granules (EC50 = the concentration producing a half maximal condensation, 9 pM). The response was rapid, reached a maximum (approximately 80% decrease in pigmented area) by 10 min, and was reversible after removal of aMT from the culture medium. 4. Aggregation to aMT was blocked by treating melanophores with pertussis toxin (1 microgram ml-1, 7 h) indicating a role for a guanosine 5' triphosphate (GTP)-binding protein in transducing the aMT receptor signal. 5. Structure-activity studies indicated that analogues of aMT lacking a side-chain N-acyl substituent (5-methoxytryptamine, MT) or a group at the 5-position of the indole ring (N-acetyltryptamine, aT) were unable to induce pigment aggregation (EC50 greater than 10 microM). 6. Lengthening the side-chain N-acyl group (N-propionyl, N-butanoyl) was tolerated to some degree but eventually (N-valeroyl and larger) activity diminished. Of the 5-position analogues tested 5-methoxy (aMT) was by far the most potent. 7. Halogen substitution in the 6-position of the indole ring led to some loss of activity as did a 6-OH substitution. The 6-OCH3 compound was inactive.8. These studies demonstrate the utility of this model in investigations of structure-activity relationships at the aMT receptor and suggest that it may be a valuable system for determining the transduction mechanisms coupled to the aMT receptor.

Animals↗

PubMed1991

The assessment of movement skill problems in 7- and 9-year-old children.

This paper is part of a project examining movement skill problems in an educational setting, and aims to provide guidelines for the assessment and management of movement problems within the primary school. The first part of the project reported here involves teachers assessing children on tasks they perform in day to day school life. A Motor Competence Checklist (MCC) was developed and tested on 350 seven- and nine-year-old children. Developmental differences were shown on every section of the MCC, and in the bottom 10 per cent of scores boys outnumbered girls by a ratio between 2 and 3 to 1. The nature of movement problems was examined by analysing profiles of children identified by the MCC, and the resulting profiles suggest there is a need to move away from viewing children with movement problems as an homogeneous group.

Child↗

PubMed1990

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland.

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.

Animals↗

PubMed1990

Pertussis toxin does not inhibit alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cyclic AMP accumulation in rat pinealocytes.

The hypothesis that Gi might be involved in the alpha 1-adrenergic, protein kinase C (PKC)-mediated amplification of beta-adrenergic cyclic AMP stimulation in rat pinealocytes was investigated. Treatment of pinealocytes with a high concentration of pertussis toxin (500 ng/ml, 18 h) almost completely (approximately 95%) inactivated two cell membrane G-proteins (kDa 40.7 and 39.8) judged by back ADP-ribosylation of pinealocyte membrane proteins. However, this treatment failed to inhibit either the beta-adrenergic (isoprenaline, ISO 10(-6) M), alpha 1-plus beta-adrenergic (noradrenaline, NA 10(-5) M) or beta-adrenergic plus 12-O-tetradecanoylphorbol 13-acetate (TPA 10(-7) M) induced stimulation of cyclic AMP or cyclic GMP. These results suggest that alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cyclic AMP and cyclic GMP does not involve a pertussis toxin-sensitive G-protein.

Adenosine Diphosphate Ribose↗

PubMed1990

Beta-adrenergic regulation of cyclic GMP in rat pinealocytes.

Stimulation of rat pinealocyte cyclic GMP by beta-adrenergic agonists (in the presence of prazosin to block alpha 1-adrenoceptors) showed an identical potency order (isoproterenol greater than epinephrine = norepinephrine much greater than salbutamol greater than terbutaline) to the stimulation of pinealocyte cyclic AMP. The increase in cyclic AMP and cyclic GMP produced by norepinephrine was inhibited by various subtype-selective beta-adrenergic antagonists with the same potency order (propranolol greater than atenolol = ICI 118,551 greater than practolol). These results indicate that pinealocytes have a single beta-adrenoceptor, of the beta 1-subtype. Activation of this beta 1-adrenoceptor mediates adrenergic stimulation of not only cyclic AMP but also cyclic GMP.

Adrenergic alpha-Agonists↗