Gottschall P E, Tatsuno I, Miyata A, Arimura A
U.S.-Japan Biomedical Research Laboratories, Tulane University, Hebert Center, Belle Chasse, Louisiana 70037.
Endocrinology. 1990 Jul;127(1):272-7. doi: 10.1210/endo-127-1-272.
A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACAP was present in two bioactive, amidated forms, PACAP27 and PACAP38 (27 and 38 amino acids, respectively), and showed a 68% sequence homology with vasoactive intestinal peptide (VIP) in the N-terminal 28 residues. PACAP38 was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells, but both peptides exhibited comparable vasodepressor activity. Thus, we sought to determine whether PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these binding sites are different from those of VIP. Binding of [125I] PACAP27 to freshly prepared rat anterior pituitary membranes in the presence and absence of 212 nM unlabeled PACAP27 was specific, saturable, and more rapid at 22 C than at 4 C. Scatchard analysis of this binding site using increasing doses of unlabeled PACAP27 revealed a single high affinity site with a Kd of 446 +/- 141 pM and a maximum number of sites of 1312 +/- 182 fmol/mg protein. These results do not exclude the possibility of a second pituitary binding site with significantly lower affinity. Unlabeled PACAP38 and PACAP38OH exhibited significantly higher affinity binding (3- to 5-fold) than PACAP27 with a similar number of pituitary sites. A variable distribution of binding sites was observed between PACAP27 and VIP when binding to different tissue membranes was measured with 125I-labeled peptides. Very high specific binding of both PACAP27 and VIP was observed in lung membranes. An almost identical relative magnitude of binding was observed between PACAP27 and VIP in lung, liver, duodenum, ovary, and thymus. However, whereas PACAP27 binding to hypothalamic and pituitary membranes was great, VIP binding to these tissues was almost absent. To determine if VIP and PACAP might share a binding site in peripheral tissues, displacement curves were generated using [125I]PACAP27 binding to lung membranes and VIP, PACAP27, and PACAP38 as unlabeled ligands. VIP was highly potent in displacing [125I] PACAP27 binding in lung membrane, and the IC50 values for all three of these peptides were between 1-10 nM. These results suggest that 1) a saturable, high affinity binding site for PACAP is present on anterior pituitary membranes; 2) PACAP27 and PACAP38, but not VIP, share this binding site in the anterior pituitary and possibly the hypothalamus; and 3) PACAP27, PACAP38, and VIP share a similar or identical binding site on lung membranes and possibly other peripheral tissues.
最近从绵羊下丘脑分离出一种新型生物活性肽，命名为PACAP（垂体腺苷酸环化酶激活多肽）。PACAP有两种生物活性酰胺化形式，即PACAP27和PACAP38（分别含27和38个氨基酸），在N端28个残基中与血管活性肠肽（VIP）有68%的序列同源性。PACAP38在刺激垂体细胞腺苷酸环化酶方面比VIP至少强1000倍，但两种肽表现出相当的血管降压活性。因此，我们试图确定PACAP是否作用于垂体前叶或其他组织中的特异性结合位点，以及这些结合位点是否与VIP的不同。在有和没有212 nM未标记PACAP27存在的情况下，[125I]PACAP27与新鲜制备的大鼠垂体前叶膜的结合是特异性的、可饱和的，并且在22℃时比在4℃时更快。使用递增剂量的未标记PACAP27对该结合位点进行Scatchard分析，发现一个单一的高亲和力位点，Kd为446±141 pM，最大位点数量为1312±182 fmol/mg蛋白。这些结果不排除存在亲和力明显较低的第二个垂体结合位点的可能性。未标记的PACAP38和PACAP38OH在垂体位点数量相似的情况下，表现出比PACAP27高得多的亲和力结合（3至5倍）。当用125I标记的肽测量与不同组织膜的结合时，观察到PACAP27和VIP之间结合位点的分布存在差异。在肺膜中观察到PACAP27和VIP都有非常高的特异性结合。在肺、肝、十二指肠、卵巢和胸腺中，PACAP27和VIP之间观察到几乎相同的相对结合强度。然而，虽然PACAP27与下丘脑和垂体膜的结合很强，但VIP与这些组织的结合几乎不存在。为了确定VIP和PACAP是否可能在外周组织中共享一个结合位点，使用[125I]PACAP27与肺膜的结合以及VIP、PACAP27和PACAP38作为未标记配体生成了置换曲线。VIP在置换肺膜中[12�I]PACAP27的结合方面非常有效，这三种肽的IC50值都在1至10 nM之间。这些结果表明：1）垂体前叶膜上存在一个可饱和的、高亲和力的PACAP结合位点；2）PACAP27和PACAP38，但不是VIP，在垂体前叶以及可能在下丘脑中共享这个结合位点；3）PACAP27、PACAP38和VIP在肺膜以及可能的其他外周组织上共享一个相似或相同的结合位点。
