Characterization of melatonin receptors in the ram pars tuberalis: influence of light

1990 ◽  
Vol 123 (5) ◽  
pp. 557-562 ◽  
Author(s):  
Jean Pelletier ◽  
Bertrand Castro ◽  
Georges Roblot ◽  
Renée Wylde ◽  
Marie-Madeleine de Reviers
Keyword(s):  
Median Eminence ◽  
Binding Sites ◽  
Melatonin Receptors ◽  
Scatchard Analysis ◽  
Pars Tuberalis

Abstract. The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20°C, but unstable at 28 or 37°C; 3. melatonin and [12 7I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 1010 l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25±0.30 vs 1.01±0.17 fmol/mm2 pars tuberalis, p<0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Identification and characterization of a corticotrophin-releasing hormone receptor in human placenta

1995 ◽  
Vol 133 (5) ◽  
pp. 591-597 ◽  
Author(s):  
Vicki L Clifton ◽  
Phillip C Owens ◽  
Phillip J Robinson ◽  
Roger Smith
Keyword(s):  
Human Placenta ◽  
Binding Sites ◽  
Hormone Receptor ◽  
Human Myometrium ◽  
Scatchard Analysis ◽  
Releasing Hormone ◽  
Rat Pituitary ◽  
Identification And Characterization ◽  
Crh Receptors

Clifton VL, Owens PC, Robinson PJ, Smith R. Identification and characterization of a corticotrophinreleasing hormone receptor in human placenta. Eur J Endocrinol 1995;133:591–7. ISSN 0804–4643 Corticotrophin-releasing hormone (CRH) causes vasodilatation in the human fetal–placental circulation and has paracrine actions in placental tissue, suggesting that CRH receptors may be present in the human placenta. We have now identified and characterized placental CRH binding sites and compared them to those described previously in human myometrium and rat pituitary. Radiolabelled ovine CRH binding to placental membranes was pH-, time-, temperature- and divalent cation-dependent and was reversible in the presence of 1 μmol/l unlabelled ovine CRH. Scatchard analysis of placentae delivered vaginally or by elective caesarean section revealed dissociation constants (Kd) of 214.5 ± 84 pmol/l (N = 8) and 45.4 ± 23.9 pmol/l (N = 9), respectively. The Kd for caesarean placental binding sites was similar to that of human myometrium (59.6 pmol/l, N = 3) and rat pituitary (82.5 pmol/l, N = 3) receptors. However, in vaginally delivered placentae the CRH binding sites had a much lower affinity (p < 0.05). The receptor densities (Bmax) of vaginally delivered and caesarean-delivered placentae were 28.6 ± 9.6 and 6.1 ± 2.8 fmol/mg, respectively (p < 0.05). Chemical cross-linking studies using disuccinimidyl suberate indicated that the molecular weight of the CRH receptor in the placenta and rat pituitary is 75 kD. We conclude that there is a high-affinity population of CRH binding sites in the human placenta that are physicochemically similar to pituitary and myometrial CRH receptors. The CRH receptor properties in the placenta change in response to labour, when CRH levels in maternal blood are highest, suggesting that placental CRH may regulate its receptor. R Smith, Endocrinology Unit, John Hunter Hospital, Locked Bag 1, Hunter Regional Mail Centre, Newcastle, NSW 2310, Australia

Characterization of [3H] desmethylimipramine binding in bovine adrenal medulla: interactions with σ- and (or) phencyclidine-receptor ligands

1992 ◽  
Vol 70 (11) ◽  
pp. 1508-1514 ◽  
Author(s):  
Cheryl Rogers ◽  
Simon Lemaire
Keyword(s):  
Adrenal Medulla ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Noradrenaline Uptake ◽  
Receptor Ligands ◽  
Mk 801 ◽  
Bovine Adrenal Medulla ◽  
Sodium Dependent ◽  
Stimulation Of

High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) σ-receptor ligands, with the following order of potency: haloperidol > rimcazole > (−)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (−)-SKF-10047. The inhibition produced by σ ligands was not attributed to stimulation of either σ1- or σ2-receptors, owing to inactivity of the selective σ-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by σ- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the σ-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and σ-ligands.Key words: desmethylimipramine, σ-receptor, phencyclidine, noradrenaline uptake, adrenal medulla.

Characterization of Melatonin Binding Sites in the Pars Tuberalis of the European Hamster

1992 ◽  
Vol 4 (2) ◽  
pp. 189-192 ◽  
Author(s):  
D. J. Skene ◽  
M. Masson-Pévet ◽  
P. Pévet
Keyword(s):  
Binding Sites ◽  
Pars Tuberalis

Melatonin Receptors in the Lamb Pars tuberalis/Median Eminence throughout the Day

1993 ◽  
Vol 58 (3) ◽  
pp. 359-365 ◽  
Author(s):  
Vincent Piketty ◽  
Jean Pelletier
Keyword(s):  
Median Eminence ◽  
Melatonin Receptors ◽  
Pars Tuberalis

Myosin VIIA is specifically associated with calmodulin and microtubule-associated protein-2B (MAP-2B)

2001 ◽  
Vol 354 (2) ◽  
pp. 267-274 ◽  
Author(s):  
Penio T. TODOROV ◽  
Rachel E. HARDISTY ◽  
Steve D. M. BROWN
Keyword(s):  
Binding Sites ◽  
Strong Association ◽  
Mouse Kidney ◽  
Scatchard Analysis ◽  
Tail Region ◽  
Immunoblotting Analysis ◽  
Kd Values ◽  
Myosin Viia

Myosin VIIA is a motor molecule with a conserved head domain and tail region unique to myosin VIIA, which probably defines its unique function in vivo. In an attempt to further characterize myosin VIIA function we set out to identify molecule(s) that specifically associate with it. We demonstrate that 17 and 55kDa proteins from mouse kidney and cochlea co-purify with myosin VIIA on affinity columns carrying immobilized anti-myosin VIIA antibody. N-terminal sequencing and immunoblotting analysis identified the 17kDa protein as calmodulin, whereas MS and immunoblotting analysis identified the 55kDa protein as microtubule-associated protein-2B (MAP-2B). Myosin VIIA can also be co-immunoprecipitated from kidney homogenate using anti-calmodulin or anti-MAP2 (recognizing isoforms 2A and 2B) antibodies, confirming the strong association between calmodulin and myosin VIIA and between MAP-2B and myosin VIIA. Myosin VIIA binds to calmodulin with an apparent Kd of 10-9 M. Scatchard analysis of the binding of myosin VIIA to MAP-2B provided evidence for two binding sites, with Kd values of 10-10 and 10-9 M, which have been mapped to medial and C-terminal tail domains of myosin VIIA. The characterization of the interaction of calmodulin and MAP-2B with myosin VIIA provides new insights into the function of myosin VIIA.

Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

Life Sciences ◽  
1991 ◽  
Vol 48 (12) ◽  
pp. 1165-1171 ◽  
Author(s):  
M.A. Lopez-Gonzalez ◽  
J.R. Calvo ◽  
A. Rubio ◽  
R. Goberna ◽  
J.M. Guerrero
Keyword(s):  
Median Eminence ◽  
Binding Sites ◽  
Harderian Gland

Melatonin receptors are present in adult rat Leydig cells and are coupled through a pertussis toxin-sensitive G-protein

1997 ◽  
Vol 136 (6) ◽  
pp. 633-639 ◽  
Author(s):  
Sandra Valenti ◽  
Massimo Giusti ◽  
Roberta Guido ◽  
Giulio Giordano
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Leydig Cells ◽  
Melatonin Receptors ◽  
Scatchard Analysis ◽  
Adult Rat ◽  
Testosterone Secretion ◽  
High Affinity Binding Site ◽  
17 Hydroxyprogesterone

Abstract Previous studies have suggested that melatonin (MLT) acts directly on rat Leydig cells by modulating androgen production. In the present study, the site of action of MLT was investigated. The binding of 2-[125I]iodomelatonin (125I-MLT; 7–240 pmol/l) to Leydig cell membrane fragments was tested in the presence or absence of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 50 μmol/l). Saturation studies and Scatchard analysis revealed the existence of a high-affinity binding site with a Bmax of 46·70± 3·50 fmol/mg protein and a Kd of 88·70±6·20 pmol/l; treatment with GTP-γ-S reduced the concentration of 125I-MLT binding sites (Bmax 34·03±4·50), while increasing the Kd to 106·5± 2·61 pmol/l. Pretreatment of the cells with pertussis toxin (PTX; 10 ng/ml for 16 h) resulted in a decreased binding of I-MLT and a lack of effect of GTP-γ-S. Moreover, the effect of MLT on testosterone secretion induced by LH (30 mIU/ml), forskolin (1 μmol/l) and LHRH (100 nmol/l) was studied after 3-h incubation of cells which had been precultured with or without PTX. The inhibition of testosterone secretion due to MLT administration was eliminated by PTX pretreatment during forskolin and LH, but not during LHRH administration. However, 17-hydroxyprogesterone levels were higher in all groups incubated in the presence of MLT, irrespective of PTX pretreatment. Our data suggest that: (a) MLT receptors are present on the membranes of adult rat Leydig cells; (b) they couple through PTX-sensitive G-protein-coupled binding sites; (c) the mechanism by which MLT blocks 17–20 desmolase enzymatic activity (thus leading to increased 17-hydroxyprogesterone levels), and testosterone secretion during LHRH stimulation is likely to depend on one or more different mechanism(s) of action. European Journal of Endocrinology 136 633–639

Demonstration and characterization of motilin-binding sites in the rabbit cerebellum

1997 ◽  
Vol 272 (5) ◽  
pp. G994-G999 ◽  
Author(s):  
I. Depoortere ◽  
T. L. Peeters
Keyword(s):  
Binding Sites ◽  
Molecular Layer ◽  
Physiological Role ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Lower Affinity ◽  
Erythromycin A ◽  
G Protein Coupled

This is the first report on central motilin receptors. Autoradiography on cerebellar slices revealed specific motilin-binding sites in the molecular layer of the cortex. Scatchard analysis of cold saturation studies showed the existence of a high-(pKd,hi = 9.07 +/- 0.09, where pKd is the negative logarithm of the dissociation constant) and a low-affinity binding site (pKd,lo = 6.56 +/- 0.09). Similar affinities were found with rabbit motilin and with the porcine (po) antagonist [Phe3, Leu13]po-motilin. Feline and canine motilin had a markedly lower affinity for the low-affinity site (pKd,lo = 5.29 and 4.58, respectively); chicken motilin had a lower affinity for both sites (pKd,hi = 8.36, pKd,lo = 3.97). Erythromycin A and its derivative N-trimethyl erythromycin A cnol ether also bound to cerebellar motilin receptors (pKd,hi = 7.29 and 8.91, respectively). Structure-activity studies with motilin fragments and the potency ranking of agonists suggest that a novel subtype receptor of motilin may exist in the brain. Guanosine 5'-O-(3-thiotriphosphate) (0.1 mM) reduced the number and the affinity for the high-affinity binding sites, which is evidence for G protein-coupled receptors. Our findings open new perspectives for the study of the physiological role of motilin.

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio)

1992 ◽  
Vol 70 (2) ◽  
pp. 268-274 ◽  
Author(s):  
Debananda Pati ◽  
Hamid R. Habibi
Keyword(s):  
Common Carp ◽  
Cyprinus Carpio ◽  
Binding Sites ◽  
Tissue Concentration ◽  
Gonadotropin Releasing Hormone ◽  
Scatchard Analysis ◽  
Single Class ◽  
Releasing Hormone ◽  
Carp Cyprinus Carpio

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 °C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0–40.0 μg membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10−6 M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-Trp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites. sGnRH-A and cGnRH-II were found to bind with greater affinities than sGnRH and GnRH-ANT to the carp ovarian binding sites. These results provide for the first time characterization of GnRH binding sites in the ovary of a teleost species, Cyprinus carpio.Key words: gonadotropin-releasing hormone, luteinizing hormone releasing hormone, receptor, ovary, carp, Cyprinus carpio.

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