Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila
Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila
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DOI：
10.1152/ajpcell.00123.2003
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发表时间：
2003-11-01
影响因子：
5.5
通讯作者：
Davies, SA
中科院分区：
文献类型：
--
作者：
Broderick, KE;MacPherson, MR;Davies, SA
一氧化氮(NO)和3‘，5’-环磷酸鸟苷(CGMP)信号转导调节果蝇体内液体转运。表达可诱导的编码果蝇一氧化氮合酶(DNOS)的转基因基因可增加Malighian(肾)小管的NOS活性和I型(主细胞)和II型(星状)细胞的DNOS蛋白。然而，cGMP含量只在主细胞中增加。在磷酸二酯酶(PDE)抑制剂扎匹司特存在的情况下，DNOS的过表达导致液体基础转运速率增加。直接检测野生型和DNOS转基因株系中管状cGMP水解型磷酸二酯酶(CG-PDE)的活性表明，CG-PDE的活性对扎普司特敏感，并且在DNOS诱导下活性升高。扎匹司特处理增加了肾小管中cGMP的含量，特别是在主细胞的顶端区域，这表明扎普司特敏感的CG-PDE定位于这些区域。通过靶向的aequorin转基因，在体内评估了激活的NO/cGMP和钙信号之间的潜在串扰。激活的DNOS信号不能单独改变神经肽(CAP(2b))或cGMP诱导的胞浆钙水平的增加。然而，在扎匹司特存在的情况下，CAP(2b)和cGMP刺激的钙水平在DNOS过度表达时都被增强。钙通道阻滞剂维拉帕米的使用，取消了扎普司特诱导的DNOS高表达小管的转运表型。通过对NO/cGMP信号通路的分子遗传学干预，发现细胞特异性CG-PDE通过直接影响细胞内cGMP的浓度和定位以及与钙信号机制的相互作用，在调节液体转运马氏管的平衡中发挥关键作用。
Signaling by nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) modulates fluid transport in Drosophila melanogaster. Expression of an inducible transgene encoding Drosophila NO synthase (dNOS) increases both NOS activity in Malpighian (renal) tubules and DNOS protein in both type I (principal) and type II (stellate) cells. However, cGMP content is increased only in principal cells. DNOS overexpression results in elevated basal rates of fluid transport in the presence of the phosphodiesterase (PDE) inhibitor, Zaprinast. Direct assay of tubule cGMP-hydrolyzing phosphodiesterase (cG-PDE) activity in wild-type and dNOS transgenic lines shows that cG-PDE activity is Zaprinast sensitive and is elevated upon dNOS induction. Zaprinast treatment increases cGMP content in tubules, particularly at the apical regions of principal cells, suggesting localization of Zaprinast-sensitive cG-PDE to these areas. Potential cross talk between activated NO/cGMP and calcium signaling was assessed in vivo with a targeted aequorin transgene. Activated DNOS signaling alone does not modify either neuropeptide (CAP(2b))- or cGMP-induced increases in cytosolic calcium levels. However, in the presence of Zaprinast, both CAP(2b)- and cGMP-stimulated calcium levels are potentiated upon DNOS overexpression. Use of the calcium channel blocker, verapamil, abolishes the Zaprinast-induced transport phenotype in dNOS-overexpressing tubules. Molecular genetic intervention in the NO/cGMP signaling pathway has uncovered a pivotal role for cell-specific cG-PDE in regulating the poise of the fluid transporting Malpighian tubule via direct effects on intracellular cGMP concentration and localization and via interactions with calcium signaling mechanisms.