Geng Y, Zhou L, Thompson W J, Lotz M
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 1998 Oct 16;273(42):27484-91. doi: 10.1074/jbc.273.42.27484.
This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.
本研究探讨了鸟苷酸环化酶（GC）和磷酸二酯酶（PDE）在白细胞介素（IL）-1激活人关节软骨细胞中的作用。GC抑制剂LY83583和亚甲蓝剂量依赖性地抑制IL-1诱导的一氧化氮（NO）生成、诱导型NO合酶（iNOS）蛋白及mRNA表达。GC抑制的这些作用与IL-1快速诱导cGMP一致，cGMP在5分钟后达到最高水平。GC抑制剂的作用具有选择性，因为它们不降低IL-1诱导的环氧化酶II蛋白和mRNA。可溶性GC特异性抑制剂不影响IL-1诱导的NO生成，可溶性GC激活剂也不诱导NO。然而，颗粒性GC激活剂心房利钠肽（ANP）和C型利钠肽（CNP）可诱导iNOS mRNA表达，表明参与iNOS诱导的是颗粒性而非可溶性鸟苷酸环化酶。cGMP的缓慢水解类似物8-溴-cGMP可诱导iNOS mRNA表达和NO生成，但不可水解类似物二丁酰cGMP则无此作用，提示介导cGMP作用的是PDE而非cGMP依赖性蛋白激酶。软骨细胞含有广泛的cGMP PDE活性。其具有PDE5的生化特征及与PDE5一致的抑制剂谱。此外，非亚型特异性PDE抑制剂异丁基甲基黄嘌呤（IBMX）和PDE5特异性抑制剂可抑制IL-1诱导的NO释放及iNOS mRNA表达。PDE5 mRNA在软骨细胞中组成性表达。除增加PDE5活性外，IL-1处理还使PDE5对几种药理抑制剂的敏感性降低达50倍。总之，GC或PDE5抑制剂均可阻止IL-1诱导iNOS；IL-1增加了cGMP生成和水解的速率；外源性可水解PDE的cGMP类似物可诱导iNOS和NO。这些结果表明，cGMP代谢通量增加足以诱导iNOS，且IL-1通过增加偶联的cGMP合成和水解诱导iNOS表达需要GC和PDE5活性。
