Miguel A. Lopez-Gonzalez

Interaction of melatonin with human lymphocytes : evidence for binding sites coupled to potentiation of cyclic AMP stimulated by vasoactive intestinal peptide and activation of cyclic GMP

Abstract: Melatonin binding sites were characterized in human blood lymphocytes. The specific binding 2‐[125I]iodomelatonin ([125I]MEL) to human lymphocytes was dependent on time and temperature, stability, saturation, and reversibility. Moreover, guanine nucleotides decreased the specific binding of [125I]MEL to crude membranes of human lymphocytes, suggesting the coupling of these binding sites to a guanosine nucleotide binding regulatory protein(s). In competition studies, the specific binding of [125I]MEL to lymphocytes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that data were compatible with the existence of two classes of binding sites: a high‐affinity site with a Kd of 5.20 ± 0.79 nM and a binding capacity of 50.6 ± 11.0 fmol/107 cells, and a low‐affinity site with a Kd of 208.5 ± 50.2 nM and a binding capacity of 2691 ± 265 fmol/107 cells. However, concentration‐dependent binding of [125I]MEL to lymphocytes was saturable and resulted in a linear Scatchard plot, suggesting binding to a single class of binding sites. The Kd for the single site was 1.02 ± 0.34 nM with a binding capacity of 10.1 ± 1.6 fmol/107 cells. Their affinities closely correlated with the production of cyclic nucleotides, suggesting a physiological role for the melatonin binding sites. Thus, melatonin potentiated the effect of vasoactive intestinal peptide (VIP) on cyclic AMP production (ED50= 1.9 nM) and stimulated cyclic GMP accumulation (ED50= 125 nM). Results demonstrate the existence of two binding sites for [125I]MEL in human blood lymphocytes, with a high‐affinity binding site coupled to the potentiation of the effect of VIP on cyclic AMP production and a low‐affinity binding site coupled to activation of cyclic GMP production.

Ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants.

The mechanism of the ototoxicity caused by cisplatin is based in the generation of reactive oxygen species, which interferes with the antioxidant protection of the organ of Corti. Conversely, the protection of the cochlea with antioxidants ameliorates the ototoxicity by cisplatin. The ototoxicity produced by cisplatin can be reversible or persistent, depending on the age of the patient, cumulative doses, number of chemotherapy cycles, history of noise exposure, and deteriorating renal function. We have obtained in rats an ototoxic chart utilizing cisplatin (10 mg/kg body weight injected intraperitoneally, once only). Together with this treatment, the animals were treated with melatonin in the drinking water (10 mg/L) or injected subcutaneously (250 μg), and with an antioxidant mixture, injected subcutaneously, composed of 0.25 mg alpha‐tocopherol acid succinate, 3 mg ascorbic acid, 1 mg glutathione, and 60 mg N‐acetylcysteine. The distortion product otoacoustic emissions were determined for a prolonged period of time for each animal. The ototoxicity produced by cisplatin was maximal from days 7 to 10 post‐treatment, returning to normal values in a month. When melatonin and the antioxidant mixture were present, the recovery was between days 10 and 15 post‐treatment, independent of the means of administration of the pineal product. We conclude that the ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants.

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

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using [125I]melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37 degrees C after 30-60 min incubation. Binding was also dependent on protein concentration (up to 1.5 mg/ml). The specific binding of [125I]melatonin was saturable, exhibiting only one class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of [125I]melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the [125I]melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.

Specific binding of 2-[125I]melatonin by partially purified membranes of rat thymus

Melatonin binding sites were characterized in partially purified rat thymus membranes. The specific binding of 2-[125I]iodomelatonin ([125I]MEL) to thymus membranes was dependent on time and temperature, stable, saturable, and reversible. Concentration-dependent binding of [125I]MEL to thymus membranes was saturable and resulted in a linear Scatchard plot, suggesting binding to a single class of binding sites. The Kd for this single site was 0.47 nM with a binding capacity of 1.01 pM. In competition studies, the specific binding of [125I]MEL to thymus membranes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that, unlike in saturation studies with [125I]MEL, data were compatible with the existence of two classes of binding sites: a high-affinity site with a Kd of 1.72 +/- 0.25 nM and a binding capacity of 1.40 +/- 0.18 pM, and a low-affinity site with a Kd of 1226 +/- 325 nM and a binding capacity of 460 +/- 87 pM. Interestingly, Kd and BC values of the high-affinity binding site described by competition studies are similar to those obtained by saturation studies with [125I]MEL. Binding of [125I]MEL to thymus membranes was specific as indicated by the fact no other melatonin precursor or derivative was as potent as melatonin in inhibiting the binding of [125I]MEL to membranes. Results strongly suggest that melatonin is involved in regulation of thymus activity.

Synergistic action of melatonin and vasoactive intestinal peptide in stimulating cyclic AMP production in human lymphocytes

Abstract: In the present study we investigated the synergistic effect of melatonin and vasoactive intestinal peptide (VIP) on cyclic AMP production in human blood lymphocytes. As shown by our group previously, VIP alone behaved as a potent activator of cyclic AMP production in human lymphocytes. On the other hand, melatonin alone did not affect the intracellular levels of cyclic nucleotide at any time or dose studied. However, when cells were incubated with melatonin plus VIP, melatonin potentiated the effect of the peptide. This effect can be observed in the presence of physiological doses of both melatonin (10–100 pM) and VIP (1–100 pM). The effect is specific for VIP because with other peptides belonging to the secretin‐VIP family the effect was not observed. Results suggest that melatonin, in conjunction with VIP, may directly participate in the regulation of immune function in the human.

Binding of 2-[125I]melatonin by rat thymus membranes during postnatal development

Previously, we have demonstrated the presence of melatonin binding sites in thymus membranes of adult rats. In this paper, we show that the binding of melatonin by thymus membranes changes during postnatal development. Maximum binding was observed in newborn rats; thereafter, binding decreased progressively during the first weeks of life and exhibited the lowest values in adult animals. Stoichiometric studies showed that the decrease in melatonin binding was due to changes in the binding capacity (2.5-fold) rather than to changes in the affinity of the receptor for the ligand. The results suggest a physiological role of melatonin in regulating thymus activity early during postnatal development.

Ototoxicity caused by aminoglycosides is ameliorated by melatonin without interfering with the antibiotic capacity of the drugs

The production of free radicals seems to be involved in the mechanisms of ototoxicity. Aminoglycosides produce ototoxicity, which can be determined through distortion product otoacoustic emissions (OAEs) that measure the activity of the outer hair cells of the organ of Corti. An ototoxic chart was obtained in rats using gentamicin or tobramycin. Together with this treatment, the animals ingested melatonin in the drinking water, or melatonin was injected subcutaneously or intramuscularly. The distortion product OAEs were determined over a prolonged period of time for each of the groups. The effect of melatonin on the antibiotic capacity of the aminoglycosides used was also studied. Antibiograms inoculated with Escherichia coli or Pseudomonas aeruginosa and treated with gentamicin or tobramycin in the presence or absence of melatonin at quantities from pharmacological to physiological doses were performed. The ototoxicity produced by gentamicin and tobramycin was maximal from days 3 to 5 post‐treatment, returning to normal values in 2 wk. When melatonin was present, the recovery was at day 5 post‐treatment, independently of the means of administration of the pineal product. The antibiograms showed that melatonin had no effect on the antibiotic capacity. It is concluded that the ototoxicity caused by gentamicin and tobramycin is ameliorated by melatonin and that the pineal hormone does not interfere with the antibiotic capacity of these antibiotics.

Melatonin binding sites in the Harderian gland of Syrian hamsters: Sexual differences and effect of castration

Abstract: The presence of specific melatonin binding sites in the Harderian gland of Syrian hamsters was studied using [125I]melatonin. Saturation binding experiments conducted with [125I] melatonin at 37°C using Harderian glands of both male and female Syrian hamsters revealed a single nanomolar‐affinity site. The dissociation constants (Kd) were 6.47 and 6.94 nM for males and females, respectively. The concentration of the binding sites was 7.58 fmol/mg protein for males and 13.50 fmol/mg protein for females. Castration of male hamsters resulted in a significant increase in [125I] melatonin binding sites while chronic melatonin administration did not modify the binding properties. The results confirm the presence of melatonin binding sites in the Harderian glands of rodents. The gender‐associated differences found together with the effects of castration in male hamsters suggest an androgenic control in [125I] melatonin binding sites of the Syrian hamster Harderian gland.

Melatonin potentiates cyclic AMP production stimulated by vasoactive intestinal peptide in human lymphocytes

The present paper demonstrates the effect of melatonin on cyclic AMP production in human lymphocytes from peripheral blood. Melatonin by itself did not influence cyclic AMP accumulation in these cells at any dose studied; however, the drug potentiated the effect of vasoactive intestinal peptide (VIP) on the cyclic nucleotide production. In the presence of physiological concentrations of VIP (either 1, 10 or 100 pM), melatonin potentiated cyclic AMP production. However, at high doses of VIP (either 1, 10 or 100 nM), melatonin exhibited no such effect. The results suggest that human lymphocytes are a target for melatonin and that it may participate, jointly with VIP, in the regulation of immune function.

Characteristics of Receptors for VIP in Rat Peritoneal Macrophage Membranes

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with Kd values of 0.60 +/- 0.08 and 275 +/- 39 nM and binding capacities of 580 +/- 71 and 72,500 +/- 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC50 = 1 nM) > rGRF (IC50 = 13 nM) > PHI (IC50 = 421 nM) >> secretin. Glucagon, somatostatin, insulin octapeptide of cholecystokinin [CCK(26-33)], and pancreastatin were ineffective at concentrations up to 1 microM. Binding of [125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [125I]VIP-protein complexes of M(r) 55,000 +/- 1700, 35,000 +/- 900, and 22,000 +/- 500.