Leiba H, Garty N B, Schmidt-Sole J, Piterman O, Azrad A, Salomon Y
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Eur J Pharmacol. 1990 May 31;181(1-2):71-82. doi: 10.1016/0014-2999(90)90246-3.
The melanocortin receptors in intraorbital and extraorbital rat lacrimal glands were studied with [125I][Nle4,D-Phe7]alpha MSH as radioligand and with several unlabeled melanocortin peptides. The pharmacological properties of the melanocortin receptor in both tissues appeared to be essentially identical. Receptor binding was studied in a membrane fraction sedimented at 12,000-100,000 X g, establishing for [125I][Nle4,D-Phe7]alpha MSH a KD of 0.76 and 2.2 nM for the intra- and extraorbital glands, respectively. Binding of the radioligand was competitively inhibited by alpha MSH (alpha-melanocyte stimulating hormone) and ACTH-(1-24) with IC50 values in the submicromolar range. MSH binding in both tissues was abolished by EGTA and was increased dose dependently with elevation of free Ca2+ ion concentration. The half-maximal effect on MSH binding was obtained around 200 microM Ca2+ and maximal binding was reached at nearly 2 mM free Ca2+ in membrane preparations from both tissues. The calmodulin-binding peptides, melittin, mastoparan and M5, the latter being the 18-amino acid synthetic analogue of the C-terminal calmodulin-binding domain of myosin light chain kinase, inhibited MSH binding in the concentration range of 1-20 microM. Macroscopic autoradiographic analysis of cryosections prepared from either lacrimal gland to which [125I][Nle4,D-Phe7]alpha MSH was subsequently bound, showed the melanocortin receptor to be uniformly distributed within the acinar lobes. At the microscopic level, MSH was found to be associated with the acinar cells, primarily at the basal perinuclear region. Peroxidase secretion from extraorbital lacrimal slices was stimulated by MSH, epinephrine and carbamylcholine to a similar extent. The response of the tissue to stimulation by MSH was however not blocked by alpha/beta-adrenoceptor blockers or by atropine, suggesting that MSH acts as a primary secretagogue in this tissue. Thus, this system seems to be uniquely suited to serve as a model for the study of both the molecular and pharmacological details of the action of MSH and other melanocortins in a non-melanogenic tissue.
以[125I][Nle4,D-Phe7]α-MSH作为放射性配体，并结合几种未标记的促黑素细胞激素肽，对大鼠眶内和眶外泪腺中的促黑素细胞激素受体进行了研究。两种组织中促黑素细胞激素受体的药理学特性似乎基本相同。在以12,000 - 100,000×g离心沉淀的膜组分中研究受体结合，结果表明，对于[125I][Nle4,D-Phe7]α-MSH，眶内和眶外泪腺的解离常数（KD）分别为0.76 nM和2.2 nM。放射性配体的结合受到α-MSH（α-黑素细胞刺激素）和促肾上腺皮质激素（1 - 24）的竞争性抑制，其半数抑制浓度（IC50）值在亚微摩尔范围内。两种组织中的促黑素细胞激素结合均被乙二醇双四乙酸（EGTA）消除，并且随着游离Ca2+离子浓度的升高呈剂量依赖性增加。在两种组织的膜制剂中，游离Ca2+浓度约为200μM时对促黑素细胞激素结合产生半数最大效应，游离Ca2+浓度接近2 mM时达到最大结合。钙调蛋白结合肽、蜂毒肽、马斯托帕兰和M5（后者是肌球蛋白轻链激酶C末端钙调蛋白结合结构域的18个氨基酸合成类似物）在1 - 20μM浓度范围内抑制促黑素细胞激素结合。对随后结合了[125I][Nle4,D-Phe7]α-MSH的任一泪腺制备的冷冻切片进行宏观放射自显影分析，结果显示促黑素细胞激素受体在腺泡叶内均匀分布。在显微镜水平上，发现促黑素细胞激素与腺泡细胞相关，主要位于核周基底部区域。眶外泪腺切片中的过氧化物酶分泌受到促黑素细胞激素、肾上腺素和卡巴胆碱的刺激程度相似。然而，该组织对促黑素细胞激素刺激的反应并未被α/β-肾上腺素能受体阻滞剂或阿托品阻断，这表明促黑素细胞激素在该组织中作为主要促分泌素发挥作用。因此，该系统似乎特别适合作为研究促黑素细胞激素和其他促黑素在非黑素生成组织中作用的分子和药理学细节的模型。
