Yao Jian, Hiramatsu Nobuhiko, Zhu Ying, Morioka Tetsuo, Takeda Masayuki, Oite Takashi, Kitamura Masanori
Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan.
J Am Soc Nephrol. 2005 Jan;16(1):58-67. doi: 10.1681/ASN.2004060453. Epub 2004 Nov 10.
This study investigated a potential role of nitric oxide (NO) in the regulation of gap junctional intercellular communication (GJIC). Incubation of mesangial cells (MC) with NO donor S-nitroso-N-acetylpenicillamine (SNAP) enhanced both basal and 8-bromo-cAMP-stimulated GJIC as well as expression of gap junction protein connexin43 (Cx43). This potentiating action of SNAP on Cx43 expression was mimicked by two other NO donors and significantly blocked by soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-1. Guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-bromo-cGMP exerted an effect similar to NO, whereas another cGMP analogue, 8-pCPT-cGMP, which selectively activates cGMP-dependent kinase without affecting cGMP-inhibited phosphodiesterase (PDE3), had no effect. Moreover, the synergistic action of NO on Cx43 expression was completely prevented by protein kinase A inhibitor H89 but not by cGMP-dependent kinase inhibitor Rp-8-Br-PET-cGMP. These results suggested a possible involvement of NO-cAMP interaction via cGMP-mediated inhibition of PDE3. Indeed, PDE3 inhibitor cilostamide caused potentiation of 8-bromo-cAMP-elicited elevations of Cx43 expression that is similar to the effect of SNAP, and an elevation of intracellular cAMP was detected in SNAP-treated cells. With the use of genetically engineered reporter MC that express secreted alkaline phosphatase under the control of the cAMP response element, significant potentiation of cAMP-elicited activation of cAMP response element by SNAP was found. This effect was abrogated in the presence of PDE3 inhibitor cilostamide. Taken together, the results suggest that NO is involved in the control of GJIC and Cx43 expression. This effect of NO is due to activation of protein kinase A via cGMP-dependent inhibition of PDE3 activity.
本研究调查了一氧化氮（NO）在调节间隙连接细胞间通讯（GJIC）中的潜在作用。用NO供体S-亚硝基-N-乙酰青霉胺（SNAP）孵育系膜细胞（MC）可增强基础的和8-溴-cAMP刺激的GJIC以及间隙连接蛋白连接蛋白43（Cx43）的表达。另外两种NO供体模拟了SNAP对Cx43表达的这种增强作用，且该作用被可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]恶二唑并[4,3,-α]喹喔啉-1-1显著阻断。鸟苷3',5'-环磷酸（cGMP）类似物8-溴-cGMP发挥了与NO类似的作用，而另一种cGMP类似物8-pCPT-cGMP，其选择性激活cGMP依赖性激酶而不影响cGMP抑制的磷酸二酯酶（PDE3），则没有作用。此外，蛋白激酶A抑制剂H89完全阻止了NO对Cx43表达的协同作用，但cGMP依赖性激酶抑制剂Rp-8-Br-PET-cGMP则没有。这些结果表明，NO可能通过cGMP介导的PDE3抑制参与cAMP相互作用。事实上，PDE3抑制剂西洛他唑引起8-溴-cAMP诱导的Cx43表达升高的增强，这与SNAP的作用类似，并且在SNAP处理的细胞中检测到细胞内cAMP升高。使用在cAMP反应元件控制下表达分泌性碱性磷酸酶的基因工程报告MC，发现SNAP对cAMP诱导的cAMP反应元件激活有显著增强作用。在存在PDE3抑制剂西洛他唑的情况下，这种作用被消除。综上所述，结果表明NO参与了GJIC和Cx43表达的调控。NO的这种作用是由于通过cGMP依赖性抑制PDE3活性激活蛋白激酶A所致。
