豚鼠耳蜗中ATP对一氧化氮/环磷酸鸟苷途径的激活作用

Activating effect of ATP on NO/cGMP pathway in guinea pig cochlea

实验研究了豚鼠耳蜗中ATP和一氧化氮/环磷酸鸟苷途径(nitric oxide/cyclic guanosine monophosphate,NO/cGMP pathway)的关系。将40只耳廓反射灵敏的健康白色豚鼠随机分为5组,分别对其离体的耳蜗即刻灌流人工外淋巴基础液(artificial perilymph basic solution,APBS)以及溶于人工外淋巴基础液的ATP、一氧化氮合酶抑制剂左旋-NG-硝基精氨酸(L-NG-nitroarginine,L-NNA)+ATP、可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]草酸重氮[4,3-a]喹恶啉(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,ODQ)+ATP和A-23187(ca2+载体),收集耳蜗组织标本,利用放射免疫方法测定耳蜗组织中的cGMP的平均含量,比较各组之间耳蜗组织cGMP平均含量的差异。试验结果显示,向刚离体的耳蜗中灌流ATP和A-23187可以引起耳蜗组织中的cGMP含量升高,而灌流L-NNA和ODQ则可以抑制ATP所引起的耳蜗组织中cGMP含量的升高,提示在耳蜗组织中ATP可以通过升高细胞内Ca2+浓度的作用而激活NO/cGMP途径。从本实验结果可以提出假说:耳蜗中ATP从神经末梢释放,通过提高细胞内Ca2+的浓度,有激活NO/cGMP途径的作用,而NO/cGMP又能对ATP进行负反馈调节,两者共同调节耳蜗的生理功能,在耳蜗中存在ATP/Ca2+-NO/cGMP通路。

The present investigation was to study the relationship between ATP and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. Forty healthy purebred albino guinea pigs with sensitive pryer's reflex were randomly divided into five groups. Their cochleae were dissected and perfused immediately with different solutions. For the control group, the cochleae (group 1) were perfused with artificial perilymph basal solution ( APBS, containing 100 μmol/L dipyridamole, 100 μmol/L L-Arg and 1 mmol/L IBMX). Other groups were respectively perfused group 2 with 330 μmol/L ATP, group 3 with 100 μmol/ L L-NNA + 330 μmol/L ATP, group 4 with 10 μmol/L ODQ + 330 μmol/L ATP and group 5 with 10 μmol/L A-23187. All these reagents were freshly dissolved in artificial perilymph basal solution (APBS). The cochlear tissue specimens were collected and the average cGMP content was measured with 125I-cGMP RIA kit. The results showed that there was no significant difference in the average cochlear tissue weights a-mong different groups. The concentration of cGMP in the cochlear tissue of the groups perfused with ATP (59.541 ±8.744 fmol/mg) and A-23187 (55.416 ±7.018 fmol/mg) was significantly higher than those of the control group (30. 089 ±4. 876 fmol/mg) , the groups perfused with L-NNA + ATP (28. 761 ±5. 019 fmol/mg) and ODQ + ATP (34. 209 ?3. 658 fmol/mg). No significant difference was observed between the group perfused with ATP and the one with A-23187, as well as among the control group and the groups perfused respectively with L-NNA + ATP and ODQ + ATP. These results suggest that ATP elevated the con- centration of cGMP in cochlear tissue while administration of nonselective nitric oxide synthase inhibitor L-NNA and soluble guanylate cyclase inhibitor ODQ could prevent the increase of cGMP concentration induced by ATP. It is indicated that ATP is involved in the activation of NO/cGMP pathway by elevating Ca2 + concentration in the cytoplasm of the cochlea. In turn, NO/cGMP pathway may exert a negative action on the effects of ATP. It is suggested that there is an ATP/Ca2+ -NO/cGMP pathway in the guinea pig cochlea. ATP and NO/cGMP pathway jointly regulate the function of the cochlea.