Gutkowska J, Tremblay J, Antakly T, Meyer R, Mukaddam-Daher S, Nemer M
Institut de Recherches Cliniques de Montréal, Québec, Canada.
Endocrinology. 1993 Feb;132(2):693-700. doi: 10.1210/endo.132.2.8425488.
The atrial natriuretic peptide (ANP) gene is expressed in several extracardiac tissues where ANP is thought to be involved in autocrine or paracrine regulation. The current studies were designed to characterize the ANP system in rat ovaries. ANP content in rat ovaries was estimated by RIA to be 240 +/- 70 pg/mg protein. HPLC revealed the presence of the 28-amino acid circulating peptide as well as the 126-amino acid prohormone, suggesting that the ovaries are a site of ANP synthesis. Indeed, ANP messenger RNA was detected in this tissue by RNase mapping. ANP present in ovarian extracts displaced [125I]ANP from bovine adrenal receptors (R1 class) in a dose-dependent manner and in parallel to the synthetic peptide, indicating that it possesses biological activity. Immunocytochemical studies localized ANP to interstitial cells surrounding the follicles; weaker but specific staining was also observed in the ovum. High affinity ANP receptors (dissociation constant, 0.30 +/- 0.06 nM; maximum binding capacity, 160 +/- 40 fmol/mg protein) were identified in ovarian membranes. Unlabeled ANP but not c-atrial natriuretic factor (a specific agonist of ANP clearance receptors) competed with binding of [125I]ANP to ovarian membranes in a dose-dependent manner, suggesting that ovarian ANP receptors are predominantly of the R1 class. This was confirmed by cross-linking studies with [125I]ANP, which detected a single protein band with a molecular size of about 120 kilodaltons, corresponding to that of the guanylate cyclase-coupled R1 class of receptor. Consistent with the presence of biologically active receptors, ANP markedly enhanced cGMP accumulation (by 15-fold) in ovarian cells. The presence of both local ANP synthesis and high affinity transducing receptors in the ovaries indicates that the peptide plays a local role in ovarian growth or steroidogenesis.
心钠素（ANP）基因在几种心外组织中表达，人们认为ANP在这些组织中参与自分泌或旁分泌调节。当前的研究旨在对大鼠卵巢中的ANP系统进行特征描述。通过放射免疫分析法（RIA）估计大鼠卵巢中的ANP含量为240±70 pg/mg蛋白质。高效液相色谱法（HPLC）显示存在28个氨基酸的循环肽以及126个氨基酸的前激素，这表明卵巢是ANP合成的场所。实际上，通过核糖核酸酶图谱法在该组织中检测到了ANP信使核糖核酸。卵巢提取物中的ANP以剂量依赖性方式并与合成肽平行地从牛肾上腺受体（R1类）上置换出[125I]ANP，表明它具有生物活性。免疫细胞化学研究将ANP定位到卵泡周围的间质细胞；在卵子中也观察到较弱但特异性的染色。在卵巢膜中鉴定出高亲和力的ANP受体（解离常数，0.30±0.06 nM；最大结合容量，160±40 fmol/mg蛋白质）。未标记的ANP而非c型心钠素因子（ANP清除受体的特异性激动剂）以剂量依赖性方式与[125I]ANP与卵巢膜的结合竞争，这表明卵巢ANP受体主要是R1类。用[125I]ANP进行的交联研究证实了这一点，该研究检测到一条分子量约为120千道尔顿的单一蛋白带，与鸟苷酸环化酶偶联的R1类受体相对应。与生物活性受体的存在一致，ANP显著增强了卵巢细胞中cGMP的积累（增加了15倍）。卵巢中存在局部ANP合成和高亲和力转导受体表明该肽在卵巢生长或类固醇生成中起局部作用。
