Amiya Noriko, Amano Masafumi, Takahashi Akiyoshi, Yamanome Takeshi, Yamamori Kunio
School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan.
Gen Comp Endocrinol. 2007 Mar;151(1):135-41. doi: 10.1016/j.ygcen.2006.12.015. Epub 2006 Dec 30.
Profiles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the Japanese flounder were examined by a newly developed time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. A TR-FIA for alpha-MSH was newly developed, and its levels in the pituitary gland and plasma of Japanese flounder reared in a white or black tank for 5 months were compared. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, alpha-MSH antibodies, samples, and europium-labeled Des-Ac-alpha-MSH. The sensitivity of the assay, defined as twice the standard deviation at a zero dose, was 0.98 ng/ml (49 pg/well). The intra- and interassay coefficients of variation of the assay were 8.8% (n=8) and 17.3% (n=5), respectively, at about 50% binding. Cross-reactivities of Des-Ac-alpha-MSH and Di-Ac-alpha-MSH were about 100%. Cross-reactivities of adrenocorticotropic hormone, salmon gonadotropin-releasing hormone (sGnRH), and chicken GnRH-II were less than 0.2%, and that of melanin-concentrating hormone was less than 2.0% at 50% binding. Displacement curves of serially twofold-diluted hypothalamus extract, pituitary gland extract, and plasma extract of Japanese flounder with the assay buffer were parallel to the alpha-MSH standard curve. Moreover, displacement curves of serially twofold-diluted hypothalamus and/or pituitary gland extract of masu salmon, goldfish, red seabream, Japanese eel, tiger puffer, and barfin flounder with the assay buffer were also parallel to the alpha-MSH standard. In Japanese flounder, total immunoreactive (ir)-alpha-MSH levels in the pituitary gland were lower in the black tank, whereas those in the plasma tended to be higher in the black tank, suggesting that the synthesis and release of alpha-MSH are higher in the black tank. alpha-MSH-ir cells were detected in the pars intermedia and a small part of the pars distalis of the pituitary gland. alpha-MSH-ir cell bodies were located in the basal hypothalamus and alpha-MSH-ir fibers were distributed not only in the hypothalamus but also in the telencephalon, midbrain, cerebellum, and medulla oblongata, suggesting that alpha-MSH functions as a neuromodulator in the brain.
采用新开发的时间分辨荧光免疫分析法（TR-FIA）和免疫组织化学方法，检测了牙鲆中α-黑素细胞刺激素（α-MSH）的分布情况。新开发了一种用于α-MSH的TR-FIA，并比较了在白色或黑色水箱中饲养5个月的牙鲆垂体和血浆中α-MSH的水平。在固相二抗、α-MSH抗体、样品和铕标记的去乙酰化α-MSH之间进行了使用两种抗体的竞争分析。该分析方法的灵敏度（定义为零剂量时标准差的两倍）为0.98 ng/ml（49 pg/孔）。在约50%结合率时，该分析方法的批内和批间变异系数分别为8.8%（n=8）和17.3%（n=5）。去乙酰化α-MSH和双乙酰化α-MSH的交叉反应率约为100%。促肾上腺皮质激素、鲑鱼促性腺激素释放激素（sGnRH）和鸡GnRH-II在50%结合率时的交叉反应率小于0.2%，而黑色素浓缩激素的交叉反应率小于2.0%。牙鲆下丘脑提取物、垂体提取物和血浆提取物系列两倍稀释后的置换曲线与α-MSH标准曲线平行。此外，马苏大麻哈鱼、金鱼、真鲷、日本鳗鲡、虎河豚和条石鲷下丘脑和/或垂体提取物系列两倍稀释后的置换曲线也与α-MSH标准曲线平行。在牙鲆中，黑色水箱中垂体中的总免疫反应性（ir）-α-MSH水平较低，而血浆中的水平在黑色水箱中往往较高，这表明黑色水箱中α-MSH的合成和释放较高。在垂体中间部和远侧部的一小部分检测到了α-MSH-ir细胞。α-MSH-ir细胞体位于下丘脑基部，α-MSH-ir纤维不仅分布在下丘脑，还分布在端脑、中脑、小脑和延髓，这表明α-MSH在大脑中起神经调节剂的作用。
