guanosine-triphosphate and 2-iodomelatonin

Abstract: Melatonin receptors in the quail caecum were studied by 2[125I]iodomelatonin binding assay and the involvement of tyrosine protein kinase in the melatonin-induced contraction was explored. The binding of 2[125I]iodomelatonin in the quail caecum membrane preparations was saturable, reversible and of high affinity with an equilibrium dissociation constant (Kd) of 24.6 +/- 1.1 pm (n = 7) and a maximum number of binding sites (Bmax) of 1.95 +/- 0.09 fmol (mg/protein) (n = 7). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was: 2-iodomelatonin > melatonin > 2-phenylmelatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that ML(1) receptors are involved. The binding was inhibited by Mel1b melatonin receptor antagonists, luzindole and 4-phenyl-2-propionamidotetralin (4-P-PDOT) as well as by non-hydrolyzable analogs of GTP like GTPgammaS and Gpp(NH)p but not by adenosine nucleotides. The latter suggests that the action of melatonin on the caecum is G-protein linked. Cumulative addition of melatonin (1-300 nM) potentiated both the amplitude and frequency of spontaneous contractions in the quail caecum. The potentiation of rhythmic contractions was blocked by both luzindole and 4-P-PDOT. Antagonists of tyrosine kinase, genistein(2 microM) and erbstatin(4 microM) suppressed the modulation of spontaneous contractions by melatonin, but not inhibitors of protein kinase C (PKC) or protein kinase A (PKA). Melatonin-induced increment in spontaneous contraction was blocked by nifedipine (0.4 nM). Thus, we suggest that melatonin potentiates spontaneous contraction in the quail caecum via interacting with G-protein-coupled Mel(1b) receptor which may activate L-type Ca2+ channels by mobilizing tyrosine kinases.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Apamin; Binding, Competitive; Cecum; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Indoles; Melatonin; Muscle Contraction; Muscle, Smooth; Naphthalenes; Potassium Channels; Protein-Tyrosine Kinases; Quail; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Tetrahydronaphthalenes; Tryptamines

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.

Topics: Animals; Binding Sites; Brain; Chickens; Guanosine Triphosphate; Iodine Radioisotopes; Kinetics; Melatonin; Receptors, Cell Surface; Receptors, Melatonin; Regression Analysis; Temperature; Thermodynamics; Tryptamines

Binding of 2-[125I]iodomelatonin to 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized sites from chick forebrain was rapid. reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2-[125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 +/- 22 (n = 4) and 302 +/- 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 +/- 2.0 and 49.5 +/- 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than prazosin greater than N-acetylserotonin much greater than serotonin greater than metergoline greater than ketanserin greater than propranolol greater than phentolamine greater than cyproheptadine. Guanyl nucleotides inhibited binding of 2-[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high-affinity to a low-affinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane-bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.

Topics: Animals; Binding Sites; Brain; Cell Membrane; Chickens; Cholic Acids; Cytosol; Detergents; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Iodine Radioisotopes; Kinetics; Male; Melatonin; Receptors, Melatonin; Receptors, Neurotransmitter; Solubility

We have localized and characterized the binding of the melatonin agonist, 2-[125I]iodomelatonin, in the rat area postrema (AP), by using quantitative autoradiography in vitro. At equilibrium conditions, Scatchard analysis revealed saturable high-affinity binding to a single class of sites (Kd 45.9 +/- (SE) 6.0 pM and Bmax 30.8 +/- 4.6 fmol/mg protein, n = 4 experiments with a total of 18 rats). Melatonin and 6-hydroxymelatonin were potent displacers of 2-[125I]iodomelatonin binding in the AP (IC50 20 and 500 pM, respectively) while N-acetylserotonin exhibited only a modest potency (IC50 25 nM). Micromolar concentrations of guanine nucleotides dose-dependently and specifically inhibited agonist binding at 22 degrees C. Saturation studies revealed that this was due to a decrease in binding affinity. Divalent cations (4 mM CaCl2 or 2 mM MgCl2) had no detectable effect on the affinity of the binding site, whereas physiological concentrations of NaCl significantly decreased the binding affinity. These results demonstrate specific high-affinity binding sites for 2-[125I]iodomelatonin in the rat AP and suggest coupling of these putative receptors to guanosine nucleotide binding regulatory protein(s).

Topics: Animals; Autoradiography; Binding Sites; Binding, Competitive; Calcium Chloride; Cations, Divalent; Cerebral Ventricles; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Magnesium Chloride; Male; Melatonin; Rats; Rats, Inbred Strains; Receptors, Melatonin; Receptors, Neurotransmitter; Serotonin; Thionucleotides

Saturation binding experiments conducted with [125I]iodomelatonin at 0-4 degrees C in the Syrian hamster hypothalamus, revealed a single nanomolar-affinity site which was not affected by GTP. In contrast, incubation at 30 degrees C revealed two distinct binding sites with picomolar and nanomolar affinities, respectively. GTP caused a significant decrease in the affinity of only the picomolar site but did not alter its density; control: Kd = 43 +/- 6 pM, Bmax = 1.7 +/- 0.3 fmol/mg protein; GTP (1 mM): Kd = 250 +/- 52, Bmax = 3.9 +/- 2.6 fmol/mg protein. The foregoing indicates that the affinity of the putative melatonin receptor in the hamster hypothalamus is modulated by a regulatory G protein.

Topics: Animals; Binding Sites; Cricetinae; Guanosine Triphosphate; Hypothalamus; Iodine Radioisotopes; Kinetics; Male; Melatonin; Membranes; Mesocricetus; Nucleotides; Temperature