Ca2+, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland
Ca2+, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland
复制标题
DOI：
10.1007/s00360-004-0448-y
复制
发表时间：
2004-09
期刊：
影响因子：
--
通讯作者：
Y. Yamamoto;O. Katsumata;S. Furuyama;H. Sugiya
中科院分区：
文献类型：
--
作者：
Y. Yamamoto;O. Katsumata;S. Furuyama;H. Sugiya
一氧化氮（NO）作为哺乳动物组织中的细胞内和细胞间信号分子发挥着重要作用。在颌下腺中，NO 被认为参与分泌和血流的调节。 NO 是通过一氧化氮合酶 (NOS) 的激活而产生的。在这里，我们研究了兔颌下腺中 NOS 活性的调节。在细胞质和膜部分均检测到 NOS 活性。胞质和部分纯化的膜组分中 NOS 的特征（例如精氨酸的 Km 值以及钙调蛋白和 Ca2+ 的 EC50 值）相似。在细胞质和膜部分中均检测到与抗 nNOS 抗体交叉反应的蛋白质条带。经过 Triton X-100 处理后，膜部分 NOS 活性增加了 1.82 倍，但胞质部分 NOS 活性却没有增加。 NOS 活性被磷脂酸 (PA) 和磷脂酰肌醇 4,5-二磷酸 (PIP2) 抑制。磷脂对NOS活性的抑制作用随着Ca2+浓度的增加而减轻。这些结果表明，Ca2+-和钙调蛋白调节酶nNOS存在于胞质和膜部分，PA和PIP2通过调节兔颌下腺中Ca2+的作用来调节膜位点的NOS活性。
Nitric oxide (NO) plays an important role as an intra- and intercellular signaling molecule in mammalian tissues. In the submandibular gland, NO has been suggested to be involved in the regulation of secretion and in blood flow. NO is produced by activation of NO synthase (NOS). Here, we have investigated the regulation of NOS activity in the rabbit submandibular gland. NOS activity was detected in both the cytosolic and membrane fractions. Characteristics of NOS in the cytosolic and partially purified membrane fractions, such asKm values forl-arginine and EC50values for calmodulin and Ca2+, were similar. A protein band that cross-reacted with anti-nNOS antibody was detected in both the cytosolic and membrane fractions. The membrane-fraction NOS activity increased 1.82-fold with treatment of Triton X-100, but the cytosolic-fraction NOS activity did not. The NOS activity was inhibited by phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2). The inhibitory effects of phospholipids on the NOS activity were relieved by an increase in Ca2+concentrations. These results suggest that the Ca2+- and calmodulin-regulating enzyme nNOS occurs in cytosolic and membrane fractions, and PA and PIP2regulate the NOS activity in the membrane site by regulating the effect of Ca2+in the rabbit submandibular gland.