CHARACTERIZATION OF BINDING-SITES FOR BETA-ADRENERGIC AGONISTS AND VASOACTIVE-INTESTINAL-PEPTIDE IN THE RAT HARDERIAN-GLAND

Vasoactive intestinal peptide (VIP) receptors and P-adrenergic recepto rs were investigated in rat Harderian gland membranes using I-125-VIP and 125I-cyanopindolol (125I-CYP), respectively, as ligands. The recep tor bindings were rapid, reversible, saturable, specific, and dependen t on time, temperature, and membrane concentration. The stoichiometric data suggested the presence of two classes of VIP receptors with Kd v alues of 0.36 and 65.37 nM and binding capacities of 323 and 39,537 fm ol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments w ith several peptides structurally or not structurally related to VIP a s follows: VIP > helodermin > rGRF > PHI >> secretin. Glucagon, somato statin, insulin, and pancreastatin were ineffective at concentrations up to 1 mu M. However, the stoichiometric data suggest the presence of one class of binding sites for 125I-CYP. The Kd for the single site w as 290 pM with a binding capacity of 32 pmol/L. The pharmacological ch aracterization of 125I-CYP binding to membranes showed that only isopr oterenol, a P-adrenergic agonist, and norepinephrine, an alpha beta-ad renergic agonist, was as effective as propranolol in inhibiting 125I-C YP binding to Harderian gland membranes. However, alpha 1- and alpha 2 -adrenergic agonists and blockers such as methoxamine, prazosin, cloni dine, and yohimbine were shown to be ineffective. These results demons trate the presence of specific VIP and P-adrenergic receptors in the H arderian gland and suggest a role for VIP and beta-adrenergic agonists in the physiology of this gland. (C) 1996 Wiley-Liss, Inc.