Effect of PGE2 antagonist（AH23848）on iNOS protein degradation

• Main Authors: Yu-Sheng, 林裕盛
• Other Authors: 林庭慧
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/56721029952867702531

博士 === 中山醫學大學 === 口腔醫學研究所 === 94 === Excessive production of nitric oxide (NO) released by mesangial cells leads to renal damage and contributes to the pathogenesis of glomerulonephritis. A connection between NO and prostaglandin E2 (PGE2) produced at inflammatory sites is involved in this intricate mechanism. In the present study, we attempted to delineate the downstream signaling of PGE2 and to explore its role in modulating NO production in lipopolysaccharide and interferon-γ (LPS + IFNγ)-stimulated SV-40-transformed glomerular mesangial cells (MES-13 cells). PGE2 modulated NO production mainly through the EP4 receptor in a cAMP-dependent manner in MES-13 cells. The fact that LPS + IFNγ-induced NO production was greatly inhibited by AH23848, an EP4 antagonist and by NS-398, a cyclooxygenase 2 (COX-2)-specific inhibitor, but was only slightly enhanced by exogenous PGE2 or 11-deoxy PGE1, implies that high endogenous PGE2 levels in LPS + IFNγ-stimulated MES-13 cells caused it to have less responsiveness toward exogenous PGE2. AH23848 inhibited NO production and inducible nitric oxide synthase (iNOS) protein expression in concentration-dependent manners but was not correlated with the inhibition of iNOS mRNA expression. Further investigation indicated that AH23848 attenuated endogenous cAMP accumulation in MES-13 cells and modulated NO production through acceleration of iNOS degradation. Elevation of the cAMP level by forskolin (FK) restored the iNOS protein level during LPS + IFNγ depletion. On the other hand, no significant level of ubiquitinated iNOS protein was observed in FK-treated or untreated cells. AH23848 downregulated the iNOS protein in MES-13 cells through protein kinase A (PKA) since KT5720, a PKA-specific inhibitor, decreased NO production and reduced iNOS protein stability. The addition of either sodium orthovanadate or okadaic acid restored the iNOS protein level during LPS + IFNγ depletion. A short exposure of activated MES-13 cells to sodium orthovanadate or okadaic acid augmented iNOS activity. Recognition of the iNOS protein by a phospho-(serine/threonine) PKA substrate antibody (PSAb) and an anti-phosphotyrosine antibody (pTyr) indicated that posttranslational modifications of the iNOS protein in MES-13 cells had occurred. The results of this study led us to speculate that cAMP might regulate iNOS-stimulated NO synthesis through posttranslational mechanisms. Attenuation of cAMP signaling and subsequent alternation of the phosphorylation status of the iNOS protein may account for the effect of AH23848 in accelerating iNOS protein degradation in MES-13 cells.