Lloyd H G, Lindström K, Fredholm B B
Department of Pharmacology, Karolinska Institute, Stockholm, Sweden.
Neurochem Int. 1993 Aug;23(2):173-85. doi: 10.1016/0197-0186(93)90095-m.
In this study, the basal and evoked release of [3H]- and endogenous adenosine, inosine and hypoxanthine from rat hippocampal slices, labelled with [3H]adenine, was investigated. Evoked release was brought about by either electrical stimulation or energy depletion. The aim was to determine whether adenosine is formed intracellularly, and released as adenosine or extracellularly, from sequential extracellular hydrolysis of released ATP. All measurements were made in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit the enzyme adenosine deaminase. It was found that electrical field stimulation (5 min) increased the release of endogenous adenosine from hippocampal slices 10-fold and increased the proportion of [3H]-label associated with adenosine from approx 7% of the total released to 13% after the first stimulation and 20% after the second stimulation. Removal of oxygen and glucose from the superfusion medium (energy depletion) increased the release rate of endogenous adenosine 16-fold and increased the proportion of [3H]-label associated with [3H]adenosine from approx 10% of the total released to 50%. In order to prevent extracellular formation of adenosine, experiments were carried out in the presence of 50 microM alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase. AOPCP was found to be without effect on either the basal or evoked release of adenosine. In contrast, L-homocysteine thiolactone (0.1-1.0 mM) which was used to "trap" intracellular adenosine reduced both the basal and evoked release of adenosine by 70-85%. This effect of L-homocysteine thiolactone also occurred in the presence of adenosine uptake inhibitors. It is concluded from these results that adenosine is formed predominantly intracellularly in hippocampal slices and is released as adenosine as a result of either tissue depolarisation or energy depletion. Furthermore, the finding that during energy depletion there is a proportionally greater release of adenosine than other ATP breakdown products, such as inosine and hypoxanthine, indicates that energy depletion is both a potent and selective stimulus for adenosine formation and release.
在本研究中，对用[3H]腺嘌呤标记的大鼠海马切片中[3H] - 以及内源性腺苷、肌苷和次黄嘌呤的基础释放和诱发释放进行了研究。诱发释放通过电刺激或能量耗竭实现。目的是确定腺苷是在细胞内形成，并以腺苷形式释放，还是通过释放的ATP的细胞外顺序水解在细胞外形成并释放。所有测量均在5微摩尔红-9-(2-羟基-3-壬基)腺苷（EHNA）存在下进行，以抑制腺苷脱氨酶。发现电场刺激（5分钟）使海马切片内源性腺苷的释放增加了10倍，并使与腺苷相关的[3H]标记比例从总释放量的约7%增加到第一次刺激后的13%和第二次刺激后的20%。从灌注培养基中去除氧气和葡萄糖（能量耗竭）使内源性腺苷的释放速率增加了16倍，并使与[3H]腺苷相关的[3H]标记比例从总释放量的约10%增加到50%。为了防止腺苷在细胞外形成，实验在50微摩尔α，β-亚甲基ADP（AOPCP）存在下进行，AOPCP是一种外切5'-核苷酸酶抑制剂。发现AOPCP对腺苷的基础释放或诱发释放均无影响。相反，用于“捕获”细胞内腺苷的L-同型半胱氨酸硫内酯（0.1 - 1.0毫摩尔）使腺苷的基础释放和诱发释放均降低了70 - 85%。L-同型半胱氨酸硫内酯的这种作用在腺苷摄取抑制剂存在下也会发生。从这些结果得出结论，腺苷主要在海马切片的细胞内形成，并由于组织去极化或能量耗竭而以腺苷形式释放。此外，在能量耗竭期间腺苷的释放比其他ATP分解产物（如肌苷和次黄嘌呤）成比例地更大这一发现表明，能量耗竭是腺苷形成和释放的一种强效且选择性的刺激。
