2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38537Excessive release of nitric oxide (NO) by mesangial cells contributes to the pathogenesis of glomerulonephritis. Prostaglandin E2 (PGE2) produced at inflammatory sites regulates the release of NO. In glomerular mesangial cells (MES-13 cells), PGE2 modulated NO production mainly through EP4 receptor in a cAMP-dependent manner. AH23848, an EP4 antagonist, attenuated endogenous cAMP accumulation in MES-13 cells and inhibited NO production through acceleration of iNOS protein degradation. The effect of AH23848 was through protein kinase A (PKA) since KT5720, a PKA-specific inhibitor, reduced iNOS protein stability. Recognition of the iNOS protein by a phospho- (serine/threonine) PKA substrate antibody (PSAb) indicated posttranslational modifications of the iNOS protein in MES-13 cells. To further investigate the mechanism underlying the effect of AH23848 in accelerating iNOS protein degradation in MES-13 cells, the specific aims are as followed: 1) to investigate which degradation pathway is involved in accelerating iNOS protein degradation by AH23848 in MES-13 cells; 2) to investigate whether dephosphorylated form of iNOS is more susceptible for degradation than wild type iNOS protein; 3) to investigate whether dephosphorylated form of iNOS blocks iNOS dimer formation; 4) to investigate whether dephosphorylated form of iNOS blocks iNOS association with HSP90 and Rac2; 5) to investigate whether PGE2/cAMP/PKA signaling modulates iNOS-stimulated NO synthesis through transactivating other signaling pathways. The results of the proposed studies will enhance our understanding about the regulation of iNOS enzyme activity at the post-translational level and provide a pharmacological strategy to attenuate NO production in treating glomerulonephritis.一氧化氮在腎臟發炎反應中扮演重要角色，過量之一氧化氮生成會破壞腎臟機能，並促使腎小球腎炎之發生。許多的研究藉由調控誘髮型一氧化氮合成.來降低一氧化氮過量所造成之細胞毒害。我們的初步結果顯示，前列腺素E2抗拮劑(AH23848)可藉由減弱細胞內環腺甘酸含量及加速誘髮型一氧化氮合成.蛋白的分解，來抑制腎絲球細胞中產生的一氧化氮。前列腺素E2抗拮劑加速誘髮型一氧化氮合成.蛋白分解是透過蛋白質激.A，因為蛋白質激.A的抑制劑亦可加速誘髮型一氧化氮合成.蛋白的分解。由於實驗結果亦顯示誘髮型一氧化氮合成.可被絲氨基酸/滋利氨基酸蛋白質激.磷酸化，並且影響誘髮型一氧化氮合成.之活性。我們推測誘髮型一氧化氮合成.在後轉譯層次上所受到之調控與其蛋白質穩定度有關。在此份計劃書中，我們希望進一步來研究，一、前列腺素E2抗拮劑是經過哪一個特定之蛋白質降解路徑來加速誘髮型一氧化氮合成.蛋白的分解。二、是否去磷酸化後之誘髮型一氧化氮合成.比正常情況之誘髮型一氧化氮合成.更容易被降解。三、是否去磷酸化後之誘髮型一氧化氮合成.是因為無法形成二聚體而使其穩定度降低。四、是否去磷酸化後，使得誘髮型一氧化氮合成.與其保護蛋白之間的結合變差，而使其較易被分解。五、是否前列腺素E2可藉由活化其它訊息傳遞路徑來調控一氧化氮之生成。我們的研究結果將可對誘髮型一氧化氮合成.在後轉譯層次上所受到之調控有更深入之瞭解，並提供如何降低一氧化氮生成量來治療腎小球腎炎之藥理策略。Project