TARGET MOLECULES OF NITRIC OXIDE/CYCLIC GMP

在腮腺腺泡细胞中，分泌功能受毒蕈碱和β-肾上腺素能受体两种主要信号通路的调节。本研究通过对兔腮腺腺泡细胞NO生成的调控，寻找一氧化氮/环GMP信号通路的靶分子。在兔腮腺腺泡细胞中，毒蕈碱胆碱能激动剂甲胆碱诱导细胞内Ca^<2+>浓度升高，并诱导一氧化氮（NO）的产生。另一方面，α-和β-激动剂、P物质和VIP不能诱导Ca^<2+>的动员和NO的生成。Ca^<2+>动员剂如thapsigarin和Ca^<2+>离子载体a23187模拟了甲胆碱对NO生成的作用。去除细胞外Ca^<2+>可抑制甲基苯丙胺诱导的NO生成。免疫印迹分析表明，抗神经元型一氧化氮合成酶（NOS）的抗体与兔腮腺细胞质中的NOS发生交叉反应。免疫荧光实验表明，NOS存在于腺泡细胞的胞浆中，而在导管细胞中较少。通过Sephacel S-200、DEAE-Sephacel和2',5'-ADP-Sepharose层析，从兔腮腺细胞质部分中纯化出约8,100倍的NOS。NOS是NADPH和四羟基生物terine依赖性酶，在钙调素存在下，在生理范围内被Ca^<2+>激活。这些结果表明，一氧化氮是由一种神经元型一氧化氮的激活而产生的，这种激活受毒蕈碱受体激活诱导兔腮腺腺泡细胞内Ca^<2+>水平升高的调控。

In parotid gland acinar cells, the secretory function is regulated by two main signaling pathways via muscarinic and β-adrenergic receptors. In this study, we investigated regulation of NO generation in rabbit parotid acinar cells to look for target molecules of nitric oxide/cyclic GMP signaling. In rabbit parotid acinar cells, muscarinic cholinergic agonist methaacholine induced an increase in intracellular Ca^<2+> concetration and provoked nitric oxide (NO) generation. On the other hand, α- and β-agonists, substance P, and VIP failed to induce Ca^<2+> mobilization and NO generation. Ca^<2+> mobilizing reagents such as thapsigargin and Ca^<2+> ionophore A 23187 mimicked the effect of methacholine on NO generation. Methacholine-induced NO generation was inhibited by the removal of extracellular Ca^<2+>. The immunoblot analysis indicated that the antibody against a neuronal type of nitrci oxide synthase (NOS) cross-reacted with NOS in the cytosol of the rabbit parotid gland. The immunofluorescent experiments showed that the NOS exists in the cytosol of acinar cells but less in the ductal cells. NOS was purified apporoximately 8,100-fold from the cytosolic fraction of the rabbit parotid glands by chromatographies on Sephacel S-200, DEAE-Sephacel, and 2', 5'-ADP-Sepharose. The NOS was NADPH- and tetrahydroxybiopterine-dependent enzyme and activated by Ca^<2+> at a physiologicl range in the presence of calmodulin. These results suggest that NO is generated by the activation of a neuronal type of NOS, which is regulated by the increase in intracellular Ca^<2+> levels induced by the activation of muscarinic receptors in rabbit parotid acinar cells.