Vlajkovic S M, Thorne P R, Housley G D, Muñoz D J, Kendrick I S
Department of Physiology, The University of Auckland, New Zealand.
Hear Res. 1998 Mar;117(1-2):71-80. doi: 10.1016/s0378-5955(98)00004-5.
This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.
本研究调查了耳蜗外淋巴腔衬里组织中外核苷酸酶的特性。分离的豚鼠耳蜗的外淋巴间隙用含氧人工外淋巴（AP）以100微升/分钟的速率灌注来维持。在AP灌注后，将三磷酸腺苷（ATP）、二磷酸腺苷（ADP）或一磷酸腺苷（AMP）引入鼓阶，灌注停止2分钟以便底物与耳蜗组织孵育。从耳蜗收集的流出物通过反相高效液相色谱（RP-HPLC）分析腺嘌呤核苷酸代谢物。细胞外ATP和ADP通过钙/镁依赖性和钙/镁非依赖性酶促机制迅速且依次水解为腺苷。细胞外ATP、ADP和AMP的降解在完整组织存在的情况下发生，乳酸脱氢酶（LDH）活性有限（0 - 2.2%）证明了这一点。ATP酶活性不受细胞内ATP酶抑制剂（寡霉素、哇巴因、N - 乙基马来酰亚胺、100微摩尔叠氮化钠）和非特异性碱性磷酸酶（β - 甘油磷酸）的影响。ATP的水解受到5毫摩尔叠氮化钠、苏拉明、ATPγS、La3 + 和CTP的抑制，ADP的水解受到β,γ - 亚氨基ATP的抑制，AMP的降解受到α,β - 亚甲基ADP的抑制。外ATP酶、外ADP酶和外5'-核苷酸酶遵循米氏双曲线动力学，估计的Km值分别为2282微摩尔、6619微摩尔和881微摩尔。我们的结果表明耳蜗鼓阶内存在相当程度的外核苷酸酶活性，并支持其作为已知在耳蜗中发生的P2受体信号传导终止机制的作用。竞争图与由具有两个不同催化位点的同一种酶（外ADP二磷酸水解酶）介导的ATP和ADP降解一致。
