TARGET MOLECULES OF NITRIC OXIDE/CYCLIC GMP

在腮腺腺泡细胞中，分泌功能通过两个主要信号通路经由毒蕈碱受体和β-肾上腺素能受体来调节。在这项研究中，我们研究了在兔腮腺腺泡细胞中NO生成的调节，以寻找一氧化氮/环GMP信号转导的靶分子。在兔腮腺腺泡细胞中，毒蕈碱胆碱能激动剂乙酰甲胆碱可诱导细胞内Ca^2+浓度升高，并引起一氧化氮（NO）生成。另一方面，α-和β-激动剂、P物质和VIP不能诱导Ca^2+动员和NO产生。Ca^2+动员试剂如毒胡萝卜素和Ca^2+离子载体A 23187模拟了乙酰甲胆碱对NO生成的作用。细胞外Ca^<2+>的清除可抑制乙酰甲胆碱诱导的NO生成。免疫印迹分析表明，抗神经元型一氧化氮合酶（NOS）的抗体与兔腮腺胞液中的NOS发生交叉反应。免疫荧光实验显示，NOS主要分布于腺泡细胞胞浆中，导管细胞胞浆中的NOS含量较少。用SephacelS-200、DEAE-Sephacel和2 ′，5 ′-ADP-Sepharose柱层析，从兔腮腺胞浆中分离纯化了约8，100倍的NOS。NOS是NADPH和四羟基生物蝶呤依赖性酶，在生理范围内，当有钙调蛋白存在时，NOS被Ca^2+激活。这些结果表明，NO是由神经元型NOS的激活产生的，而NOS的激活受兔腮腺腺泡细胞中毒蕈碱受体激活诱导的细胞内Ca^2+水平升高的调节。

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.