Borst M M, Schrader J
Institute of Physiology, Düsseldorf University, FRG.
Circ Res. 1991 Mar;68(3):797-806. doi: 10.1161/01.res.68.3.797.
The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.
从心脏释放的腺嘌呤核苷酸的定量分析受到阻碍，因为它们在细胞外空间中被高活性的胞外核苷酸酶迅速脱磷酸化为腺苷。为了确定从离体Langendorff灌注的豚鼠心脏中腺嘌呤核苷酸的总释放量，通过输注α,β-亚甲基-ADP（AOPCP，50μM）有效地阻断了胞外5'-核苷酸酶。在酶促再磷酸化为ATP后，通过荧光素-荧光素酶法测量冠状静脉流出液中的腺嘌呤核苷酸。在用常氧缓冲液（95% O2，5% CO2）以恒定流速（10 ml/min）灌注的心脏中，腺嘌呤核苷酸和腺苷的释放量±SEM分别为0.06±0.01（n = 11）和0.04±0.01（n = 13）nmol/min。在AOPCP存在的情况下，腺嘌呤核苷酸的释放量增加到0.43±0.04 nmol/min（n = 9；p < 0.05），而腺苷保持不变。低氧灌注（10% O2，85% N2，5% CO2）导致腺嘌呤核苷酸释放量增加三倍，但腺苷释放量增加40倍。相比之下，全心缺血（30秒）导致腺嘌呤核苷酸和腺苷释放量上升到相似的值，分别为1.06±0.10和0.80±0.14 nmol/min（n = 9）。用异丙肾上腺素（4 nM）刺激心脏同样增加了腺嘌呤核苷酸（0.50±0.04 nmol/min）和腺苷（0.87±0.21 nmol/min）的释放量（n = 6）。为了确定从心脏释放的腺嘌呤核苷酸的细胞来源，冠状内皮腺嘌呤核苷酸池通过[3H]腺苷进行了选择性预标记。全心缺血使释放的腺嘌呤核苷酸的比放射性增加了57%。这些发现表明：1）在氧合良好的心脏中，腺嘌呤核苷酸和腺苷以相同的数量级释放；2）β-肾上腺素能刺激和缺血刺激腺嘌呤核苷酸和腺苷的释放，这两种嘌呤在流出灌注液中达到血管活性浓度；3）在低氧灌注期间，只有腺苷的释放量大大增加；4）冠状内皮优先促进缺血诱导的腺嘌呤核苷酸释放。
