Vlajkovic S M, Thorne P R, Muñoz D J, Housley G D
Department of Physiology, University of Auckland, New Zealand.
Hear Res. 1996 Sep 15;99(1-2):31-7. doi: 10.1016/s0378-5955(96)00079-2.
It has been clearly demonstrated that extracellular adenosine 5'-triphosphate (ATP) exerts a potent modulatory activity in the cochlea through its interaction with P2 purinoceptors. However, little is known regarding the metabolism of extracellular ATP in cochlear tissues via ectonucleotidases. This study provides evidence for the presence of ectonucleotidases in the perilymphatic compartment of the guinea pig cochlea. Using microperfusion, ATP (500 microM) was introduced into the cochlear perilymph through the basal turn scala tympani, and effluent was collected from the basal turn scala vestibuli. Samples were subsequently analysed for the presence of adenine metabolites using high performance liquid chromatography (HPLC). Cell viability was evaluated by the activity of the intracellular enzyme lactate dehydrogenase (LDH) in the perfusate. ATP was degraded to 122.8 +/- 9.9 microM (25.0 +/- 5.8%) during the passage through the cochlear perilymphatic compartment. Breakdown of ATP resulted in the formation of adenosine 5'-diphosphate (41.5 +/- 9.0 microM), adenosine 5'-monophosphate (201.3 +/- 15.5 microM), adenosine (108.6 +/- 8.3) and inosine (15.0 +/- 1.5 microM). The degradation of ATP was significantly (P < 0.001, Student's t-test) inhibited in the absence of divalent cations, Ca2+ and Mg2+ in the perfusate. In control experiments, no spontaneous degradation of ATP was observed in vitro. LDH activity was similar during ATP perfusions (2.9 +/- 0.9%) to control perfusions with artificial perilymph (4.2 +/- 1.0%) indicating well preserved cell integrity in the cochlear perilymphatic compartment. The degradation of extracellular ATP in the presence of intact tissues and its inhibition in the absence of divalent cations, a cofactor for ectonucleotidases, provides evidence for ectonucleotidase activity in the perilymphatic fluid space of the cochlea.
已经清楚地表明，细胞外5'-三磷酸腺苷（ATP）通过与P2嘌呤受体相互作用在耳蜗中发挥强大的调节活性。然而，关于耳蜗组织中细胞外ATP通过外核苷酸酶的代谢情况知之甚少。本研究为豚鼠耳蜗外淋巴间隙中存在外核苷酸酶提供了证据。使用微量灌注，将ATP（500微摩尔）通过鼓阶基底转引入耳蜗外淋巴，从前庭阶基底转收集流出物。随后使用高效液相色谱（HPLC）分析样品中腺嘌呤代谢物的存在情况。通过灌注液中细胞内酶乳酸脱氢酶（LDH）的活性评估细胞活力。ATP在通过耳蜗外淋巴间隙的过程中降解为122.8±9.9微摩尔（25.0±5.8%）。ATP的分解导致形成二磷酸腺苷（41.5±9.0微摩尔）、一磷酸腺苷（201.3±15.5微摩尔）、腺苷（108.6±8.3）和肌苷（15.0±1.5微摩尔）。在灌注液中不存在二价阳离子Ca2+和Mg2+时，ATP的降解受到显著抑制（P<0.001，学生t检验）。在对照实验中，体外未观察到ATP的自发降解。ATP灌注期间的LDH活性（2.9±0.9%）与人工外淋巴对照灌注期间的活性（4.2±1.0%）相似，表明耳蜗外淋巴间隙中的细胞完整性良好。在完整组织存在的情况下细胞外ATP的降解及其在不存在外核苷酸酶辅因子二价阳离子时的抑制作用，为耳蜗外淋巴液空间中的外核苷酸酶活性提供了证据。
