Guerrero J M, Osuna C, Molinero P, Caraballo M I, Harmouch A, Pozo D, Rafii-el-Idrissi M, Garcia-Macias J F, Calvo J R
Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine, Spain.
Microsc Res Tech. 1996 Jun 1;34(2):139-43. doi: 10.1002/(SICI)1097-0029(19960601)34:2<139::AID-JEMT7>3.0.CO;2-N.
Vasoactive intestinal peptide (VIP) receptors and beta-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 microM. 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 beta-adrenergic agonist, and norepinephrine, an alpha beta-adrenergic agonist, was as effective as propranolol in inhibiting 125I-CYP binding to Harderian gland membranes. However, alpha 1- and alpha 2-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 beta-adrenergic receptors in the Harderian gland and suggest a role for VIP and beta-adrenergic agonists in the physiology of this gland.
分别使用125I - 血管活性肠肽（VIP）和125I - 氰基吲哚洛尔（125I - CYP）作为配体，对大鼠哈德氏腺膜中的VIP受体和β - 肾上腺素能受体进行了研究。受体结合迅速、可逆、可饱和、具有特异性，且依赖于时间、温度和膜浓度。化学计量学数据表明存在两类VIP受体，其解离常数（Kd）值分别为0.36和65.37 nM，结合容量分别为323和39537 fmol VIP/mg蛋白。如用几种与VIP在结构上或非结构上相关的肽进行竞争置换实验所示，这种相互作用表现出高度特异性：VIP＞海洛德明＞大鼠生长激素释放因子＞胰高血糖素样肽＞＞促胰液素。胰高血糖素、生长抑素、胰岛素和胰抑素在浓度高达1μM时无效。然而，化学计量学数据表明存在一类125I - CYP的结合位点。单个位点的Kd为290 pM，结合容量为32 pmol/L。125I - CYP与膜结合的药理学特征表明，只有β - 肾上腺素能激动剂异丙肾上腺素和αβ - 肾上腺素能激动剂去甲肾上腺素在抑制125I - CYP与哈德氏腺膜结合方面与普萘洛尔一样有效。然而，α1 - 和α2 - 肾上腺素能激动剂及阻滞剂，如甲氧明、哌唑嗪、可乐定和育亨宾被证明无效。这些结果证明了哈德氏腺中存在特异性VIP和β - 肾上腺素能受体，并提示VIP和β - 肾上腺素能激动剂在该腺体的生理学中发挥作用。
