Binding of [125I]iodocyanopindolol by rat Harderian gland crude membranes: Kinetic characteristics and day—night variations

Abstract

The Harderian glands are innervated by sympathetic fibers originating in the superior cervical ganglia. The aim of this study is to characterize the β-adrenergic receptors in the rat Harderian gland. The characteristics of β-adrenergic receptors were determined in crude membrane preparations from rat Harderian gland, using [125I]iodocyanopindolol ([125I]CYP) as radioligand. The binding of the ligand to the receptor is rapid, reversible, saturable, specific and dependent on time, temperature and membrane concentration. At 30 °C, stoichiometric data suggest the presence of one binding site with a Kd value of 0.29 nM and Bmax of 32 pmol/L. The interaction shows a high degree of specificity for β-adrenergic agonists and blockers, as suggested by competitive displacement experiments with isoproterenol (IC50=19.1 nM), propranolol (IC50=28.1 nM), and norepinephrine (IC50=96.3 nM). Clonidine, yohimbine, methoxamine, and prazosin are ineffective at concentrations up to 1 μM. In the other hand, binding of [125I]CYP by Harderian gland membranes exhibits day—night variations. Binding values are low during the daytime and increase progressively late in the evening to reach a maximum at 2200 h (2 h after the onset of dark period), but decreased to the end of the dark period (0600 h). In conclusion, the results presented in this paper show the functional and pharmacological characterization of β-adrenergic receptors in the rat Harderian gland. This neurotransmitter may play a physiological role at this level regulating, at least, processes such as a thyroid hormone metabolism.