Lee Nikki P Y, Cheng C Yan
Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021, USA.
Endocrinology. 2003 Jul;144(7):3114-29. doi: 10.1210/en.2002-0167.
Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.
一氧化氮（NO）合酶（NOS）催化L-精氨酸氧化生成NO。NO在调节多种生理功能中起关键作用，可能包括通过其对环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）的影响来调节连接动态，而cAMP和cGMP是已知的紧密连接（TJ）动态调节因子。尽管在睾丸中发现了诱导型NOS（iNOS）和内皮型NOS（eNOS），并且它们与精子发生的调节有关，但它们在TJ动态中的作用（如果有的话）尚不清楚。当支持细胞以0.5 - 1.2×10⁶个细胞/cm²的密度接种在基质胶包被的培养皿或双室装置上培养时，通过量化跨细胞上皮的跨上皮电阻来评估屏障功能时，形成了功能性TJ屏障。结果表明，TJ屏障的组装与iNOS和eNOS水平的显著下降相关，这似乎表明它们产生的NO可能会干扰TJ的组装。为了进一步证实NOS在体外对TJ屏障功能的作用，在TJ组装过程中向支持细胞培养物中添加了锌（II）原卟啉-IX（ZnPP），它是一种NOS抑制剂和可溶性鸟苷酸环化酶抑制剂。事实上，发现ZnPP可能通过诱导TJ相关蛋白（如闭合蛋白）的产生来促进支持细胞TJ屏障的组装和维持。随后通过免疫沉淀和免疫印迹进行的研究表明，iNOS和eNOS在结构上与TJ整合膜蛋白（如闭合蛋白）以及细胞骨架蛋白（如肌动蛋白、波形蛋白和α-微管蛋白）相连。当对这些用ZnPP处理的样品中的cAMP和cGMP水平进行量化时，确实检测到ZnPP诱导细胞内cGMP水平降低，但cAMP水平未降低。此外，8-溴-cGMP，一种细胞膜可渗透的cGMP类似物，也能像8-溴-cAMP一样剂量依赖性地干扰TJ屏障。蛋白激酶G的特异性抑制剂KT-5823被证明能促进支持细胞TJ屏障的组装。已知会干扰支持细胞TJ屏障的细胞因子，如转化生长因子-β（TGF-β）和肿瘤坏死因子-α（TNF-α），在体外也被证明能剂量依赖性地刺激支持细胞iNOS和eNOS的表达。总的来说，这些结果表明NOS是睾丸中TJ动态的重要生理调节因子，通过NO/可溶性鸟苷酸环化酶/cGMP/蛋白激酶G信号通路发挥作用。
