Kurhaliuk N M, Serebrovs'ka T V, Koliesnikova Ie E
Ivan Franko Lviv National University, Ukraine.
Ukr Biokhim Zh (1999). 2002 Nov-Dec;74(6):114-9.
It is known that NO-dependent mechanisms are involved in mitochondrial adaptive reactions to different factors. The object of this study was to investigate the role of cholino- and adrenoreceptors in NO-dependent reactions of rat liver mitochondria to acute hypoxia (AH) and intermittent hypoxic training (IHT). Eight groups of Wistar male rats participated in the study. Animals of Gr. I underwent daily i.p. saline injections during 14 days. Gr. II was examined after a single AH test (inhalation of 7% O2, 30 min) with the same saline treatment. Another six groups were exposed to IHT (11% O2, 15-min sessions with 15 min rest intervals, 5 times daily during 14-days), at that 15 min before every IHT session animals underwent i.p. treatment: Gr. III and IV--saline, Gr. V--L-arginine, Gr. VI--NO synthase blocker L-NNA, Gr. VII--L-arginine with alpha-, beta-adrenoblockers phentolamine and obzidane, Gr. VIII--L-arginine with M- and N-cholinoreceptor blockers athropine and benzohexonium. After IHT Gr. IV-VIII were exposed to a single AH test and decapitated just after that. In control rats AH provoked: 1) in the presence of succinate, a 33% augmentation of ADP-stimulated mitochondrial respiration (V3) with a 18% decrease of O2 uptake efficiency (ADP/O ratio); 2) in the presence of alpha-ketoglutarate, a NAD-dependent substrate, no changes in V3 were observed, also 21% augmentation of ADP/O ratio registered. These events were accompanied by 36% increase in succinate dehydrogenase (SDG) activity, two-fold augmentation of malon dialdehyde (MDA) content and 43% increase in diene conjugates (DK). IHT caused reorganization of mitochondrial energy metabolism improving the protection against acute hypoxia. A decrease by 40% in activation of mitochondrial respiration in the presence of succinate (V3--by 40% and V4--by 34%), a reduction of MDA and DK content (by 32% and 20%, respectively), an increase in SGD activity by 31% was observed in Gr. IY compared to Gr. II. Extra exogenous NO (Gr.Y) did not influence V3 and V4 in the presence of succinate, but in the presence of alpha-ketoglutarate decreased them by 9% and 29%, respectively, as well as ADP/O ratio by 28% on the background of SDG inhibition by 24% and the decrease of MDA content by 34%, that is reduced aerobic energy supply and reactive oxygen species production. L-arginine effects were abolished by L-NNA. Effects of cholinoreceptor blockers over L-arginine (Gr. VIII) resembled effects of AH: considerable activation of succinate and alpha-ketoglutarate oxidation in stage V3 by 44% and 75%, respectively, was observed which was accompanied by a decrease in ADP/O by 21% and 31%, and V3/V4 by 15% and 28%, respectively, in comparison to Gr.Y. It indicates that effects of L-arginine are mediated mainly by cholinoreceptors. The effects of adrenoreceptors blockade strengthened the combined effects of IHT with L-arginine treatment, confirming primary role of cholinoreceptors in NO-dependent mitochondrial reactions to IHT. Thus, oxygen uptake and its effective usage depend on dynamic status of adreno- and cholinoreceptors. We conclude that protective effects of the combination of IHT with NO-donor treatment under acute hypoxia are mainly realized through cholinoreceptors.
已知一氧化氮（NO）依赖的机制参与线粒体对不同因素的适应性反应。本研究的目的是探讨胆碱能和肾上腺素能受体在大鼠肝线粒体对急性缺氧（AH）和间歇性低氧训练（IHT）的NO依赖反应中的作用。八组Wistar雄性大鼠参与了本研究。第一组动物在14天内每天腹腔注射生理盐水。第二组在单次AH试验（吸入7%氧气，30分钟）后，同样接受生理盐水处理并进行检测。另外六组接受IHT（11%氧气，每次训练15分钟，休息15分钟，每天5次，共14天），在每次IHT训练前15分钟，动物接受腹腔注射处理：第三组和第四组注射生理盐水，第五组注射L-精氨酸，第六组注射NO合酶阻滞剂L-NNA，第七组注射L-精氨酸与α、β肾上腺素能阻滞剂酚妥拉明和阿齐旦，第八组注射L-精氨酸与M和N胆碱能受体阻滞剂阿托品和苯六甲铵。IHT训练后，第四组至第八组接受单次AH试验，试验后立即断头处死。在对照大鼠中，AH引发：1）在琥珀酸存在的情况下，ADP刺激的线粒体呼吸（V3）增加33%，氧气摄取效率（ADP/O比值）降低18%；2）在α-酮戊二酸（一种NAD依赖的底物）存在的情况下，V3未观察到变化，ADP/O比值增加21%。这些变化伴随着琥珀酸脱氢酶（SDG）活性增加36%，丙二醛（MDA）含量增加两倍，二烯共轭物（DK）增加43%。IHT导致线粒体能量代谢重组，增强了对急性缺氧的保护作用。与第二组相比，第四组在琥珀酸存在时线粒体呼吸激活降低40%（V3降低40%，V4降低34%），MDA和DK含量降低（分别降低32%和20%），SGD活性增加31%。额外的外源性NO（第五组）在琥珀酸存在时不影响V3和V4，但在α-酮戊二酸存在时，分别使其降低9%和29%，同时在SDG抑制24%和MDA含量降低34%的背景下，ADP/O比值降低28%，即有氧能量供应和活性氧生成减少。L-NNA消除了L-精氨酸的作用。胆碱能受体阻滞剂对L-精氨酸的影响（第八组）类似于AH的影响：与第五组相比，观察到在V3阶段琥珀酸和α-酮戊二酸氧化分别显著激活44%和75%，同时ADP/O分别降低21%和31%，V3/V4分别降低15%和28%。这表明L-精氨酸的作用主要由胆碱能受体介导。肾上腺素能受体阻断的作用增强了IHT与L-精氨酸联合处理的综合效果，证实了胆碱能受体在IHT的NO依赖线粒体反应中的主要作用。因此，氧气摄取及其有效利用取决于肾上腺素能和胆碱能受体的动态状态。我们得出结论，IHT与NO供体联合处理在急性缺氧条件下的保护作用主要通过胆碱能受体实现。
