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.