Shen Jing, Harada Narinobu, Nakazawa Hiroko, Yamashita Toshio
Hearing Research Laboratory, Department of Otolaryngology, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8507, Japan.
Eur J Neurosci. 2005 Jun;21(11):2912-22. doi: 10.1111/j.1460-9568.2005.04135.x.
We recently demonstrated that extracellular adenosine 5'-triphosphate (ATP) induced nitric oxide (NO) production in the inner hair cells (IHCs) of the guinea pig cochlea, which inhibited the ATP-induced increase in the intracellular Ca(2+) concentrations (Ca(2+)) by a feedback mechanism [Shen, J., Harada, N. & Yamashita, T. (2003) Neurosci. Lett., 337, 135-138]. We herein investigated the role of the NO-cGMP pathway and neuronal NO synthase (nNOS) in the ATP-induced Ca(2+) signalling in IHCs using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye DAF-2. Fura-2 fluorescence-quenching experiments with Mn(2+) showed that ATP triggered a Mn(2+) influx. L-N(G)-nitroarginine methyl ester (L-NAME), a nonspecific NOS inhibitor, accelerated the ATP-induced Mn(2+) influx while S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, suppressed it. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase, and KT5823, an inhibitor of cGMP-dependent protein kinase, enhanced the ATP-induced Ca(2+) increase. 8-Bromoguanosine-cGMP, a membrane-permeant analogue of cGMP mimicked the effects of SNAP. Moreover, the effects of 7-nitroindazole, a selective nNOS inhibitor, mimicked the effects of L-NAME regarding both the enhancement of the ATP-induced Ca(2+) response and the attenuation of NO production. Immunofluorescent staining of nNOS using a single IHC revealed that nNOS was distributed throughout the IHCs, but enriched in the apical region of the IHCs as shown by intense staining. In conclusion, the ATP-induced Ca(2+) influx may be the principal source for nNOS activity, which may interact with P2X receptors in the apical region of IHCs. Thereafter, NO can be produced and conversely inhibits the Ca(2+) influx via the NO-cGMP-PKG pathway by a feedback mechanism.
我们最近证明，细胞外的5'-三磷酸腺苷（ATP）可诱导豚鼠耳蜗内毛细胞（IHC）产生一氧化氮（NO），通过反馈机制抑制ATP诱导的细胞内Ca(2+)浓度（[Ca(2+)]i）升高[Shen, J., Harada, N. & Yamashita, T. (2003) Neurosci. Lett., 337, 135 - 138]。在此，我们使用Ca(2+)敏感染料fura - 2和NO敏感染料DAF - 2，研究了NO - cGMP途径和神经元型一氧化氮合酶（nNOS）在ATP诱导的IHC中Ca(2+)信号传导中的作用。用Mn(2+)进行的fura - 2荧光猝灭实验表明，ATP触发了Mn(2+)内流。非特异性一氧化氮合酶抑制剂L - N(G)-硝基精氨酸甲酯（L - NAME）加速了ATP诱导的Mn(2+)内流，而NO供体S - 亚硝基 - N - 乙酰青霉胺（SNAP）则抑制了它。鸟苷酸环化酶抑制剂1H - [1,2,4]恶二唑[4,3 - a]喹喔啉 - 1 - 酮和cGMP依赖性蛋白激酶抑制剂KT5823增强了ATP诱导的[Ca(2+)]i升高。cGMP的膜渗透性类似物8 - 溴鸟苷 - cGMP模拟了SNAP的作用。此外，选择性nNOS抑制剂7 - 硝基吲唑对ATP诱导的Ca(2+)反应增强和NO产生减弱的作用类似于L - NAME。使用单个IHC对nNOS进行免疫荧光染色显示，nNOS分布于整个IHC，但在IHC的顶端区域富集，染色强烈。总之，ATP诱导的Ca(2+)内流可能是nNOS活性的主要来源，nNOS可能在IHC顶端区域与P2X受体相互作用。此后，可产生NO，反之通过反馈机制经由NO - cGMP - PKG途径抑制Ca(2+)内流。
