Kishimoto J, Tsuchiya T, Emson P C, Nakayama Y
Life Science Research Laboratories, Shiseido Research Center, Yokohama, Japan.
Brain Res. 1996 May 13;720(1-2):159-71. doi: 10.1016/0006-8993(96)00101-1.
The purpose of this study was to determine whether immobilization stress can cause changes in the enzyme activity and gene expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus, pituitary, and adrenal gland in rats. NOS enzyme activity was measured as the rate of [3H]arginine conversion to citrulline, and the level of nNOS mRNA signal was determined using in situ hybridization and image analysis. NOS-positive cells were also visualized using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) histochemistry and by immunohistochemistry using an anti-nNOS antibody. A significant increase of NOS enzyme activity in the anterior pituitary, adrenal cortex, and adrenal medulla (1.5-, 3.5-, and 2.5-fold) was observed in the stressed animals (immobilization of 6 h) as compared to non-stressed control rats. Up-regulation of nNOS mRNA expression in anterior pituitary and adrenal cortex was already detectable after stress for 2 h with 1.5- and 2-fold increase, respectively. The nNOS mRNA signals in hypothalamic paraventricular nucleus (PVN) significantly increased after the stress for 6 h. This increase in NOS enzyme activity was confirmed using NADPH-diaphorase staining and immunostaining in the PVN and adrenal cortex. An increase of NOS enzyme activity in adrenal medulla after immobilization for 6 h posited by far longer than in the adrenal cortex and anterior pituitary. The present findings suggest that psychological and/or physiological stress causes NO release in hypothalamic-pituitary-adrenal (HPA) axis and in sympatho-adrenal system. It is suggested that NO may modulate a stress-induced activation of the HPA axis and the sympatho-adrenal medullary system. The different duration of stress-induced NOS activity in HPA axis and the adrenal medulla may suggest NO synthesis is controlled by separate mechanism in the two HPA and the sympatho-adrenal systems.
本研究的目的是确定固定应激是否会导致大鼠下丘脑、垂体和肾上腺中神经元型一氧化氮合酶（nNOS）的酶活性和基因表达发生变化。NOS酶活性通过[3H]精氨酸转化为瓜氨酸的速率来测定，nNOS mRNA信号水平则使用原位杂交和图像分析来确定。NOS阳性细胞也通过烟酰胺腺嘌呤二核苷酸磷酸黄递酶（NADPH-黄递酶）组织化学以及使用抗nNOS抗体的免疫组织化学进行可视化。与未应激的对照大鼠相比，在应激动物（固定6小时）中观察到垂体前叶、肾上腺皮质和肾上腺髓质中的NOS酶活性显著增加（分别为1.5倍、3.5倍和2.5倍）。应激2小时后，垂体前叶和肾上腺皮质中nNOS mRNA表达上调已可检测到，分别增加了1.5倍和2倍。应激6小时后，下丘脑室旁核（PVN）中的nNOS mRNA信号显著增加。PVN和肾上腺皮质中NOS酶活性的增加通过NADPH-黄递酶染色和免疫染色得到证实。固定6小时后，肾上腺髓质中NOS酶活性的增加持续时间远比肾上腺皮质和垂体前叶长得多。目前的研究结果表明，心理和/或生理应激会导致下丘脑-垂体-肾上腺（HPA）轴和交感-肾上腺系统中一氧化氮（NO）的释放。提示NO可能调节应激诱导的HPA轴和交感-肾上腺髓质系统的激活。HPA轴和肾上腺髓质中应激诱导的NOS活性的不同持续时间可能表明，NO合成在两个HPA和交感-肾上腺系统中受不同机制控制。
