Characterization of binding sites for β-adrenergic agonists and vasoactive intestinal peptide in the rat Harderian gland

Vasoactive intestinal peptide (VIP) receptors and β-adrenergic receptors were investigated in rat Harderian gland membranes using 125I-VIP and 125I-cyanopindolol (125I-CYP), respectively, as ligands. The receptor bindings were rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 0.36 and 65.37 nM and binding capacities of 323 and 39,537 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 as follows: VIP > helodermin > rGRF > PHI >> secretin. Glucagon, somatostatin, insulin, and pancreastatin were ineffective at concentrations up to 1 μM. However, the stoichiometric data suggest the presence of one class of binding sites for 125I-CYP. The Kd for the single site was 290 pM with a binding capacity of 32 pmol/L. The pharmacological characterization of 125I-CYP binding to membranes showed that only isoproterenol, a β-adrenergic agonist, and norepinephrine, an αβ-adrenergic agonist, was as effective as propranolol in inhibiting 125I-CYP binding to Harderian gland membranes. However, α1- and α2- adrenergic agonists and blockers such as methoxamine, prazosin, clonidine, and yohimbine were shown to be ineffective. These results demonstrate the presence of specific VIP and β-adrenergic receptors in the Harderian gland and suggest a role for VIP and β-adrenergic agonists in the physiology of this gland.