Gu X H, Dillon J S, Nayler W G
Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Victoria, Australia.
Biochem Pharmacol. 1989 Jun 15;38(12):1897-907. doi: 10.1016/0006-2952(89)90487-5.
The effect of hypoxia, reoxygenation and chemically-induced high energy phosphate depletion (caused by inhibition of oxidative phosphorylation and glycolysis) on the affinity (Kd), density (Bmax) and selectivity of high affinity 1,4-dihydropyridine (DHP) calcium antagonist binding sites was studied in rat isolated cardiac membranes, using (+)[3H]PN200-110. Neither 30 nor 60 min normothermic (37 degrees) hypoxia affected either the Bmax or Kd of these sites, relative to aerobic controls. Fifteen min reoxygenation after 60, but not 30, min hypoxia reduced the density of the DHP binding sites, without altering their affinity or selectivity. Aerobic perfusion with 0.1 mM DNP (an uncoupler of oxidative phosphorylation) for 30 min at 37 degrees caused an increase in Bmax (P less than 0.05) both in the presence (48%) and absence (27%) of glucose, without any change in Kd. This increase in Bmax was attenuated during a further 30 min perfusion with DNP. Thirty min perfusion with 1.0 mM IAA and 0.1 mM DNP resulted in a significant increase (27%) in the Bmax of the DHP binding sites. A further 30 min perfusion with IAA and DNP caused the Bmax to return to control levels. The Kd was not altered under these conditions. Irrespective of the perfusion conditions, the selectivity of the binding sites was unchanged, with (+)PN200-100 greater than (-)Bay K8644 greater than (-)PN200-110 = (+)Bay K8644 in displacing bound (+)[3H]PN200-110. Under all conditions, (-)D600 always interacted allosterically with the DHP binding sites, and the binding was stimulated by d-cis diltiazem. These results show that neither hypoxia nor chemically-induced ATP depletion mimic the effect of ischaemia on cardiac DHP binding sites.
使用（+）[³H]PN200 - 110，在大鼠离体心脏膜中研究了缺氧、复氧以及化学诱导的高能磷酸耗竭（由氧化磷酸化和糖酵解抑制引起）对高亲和力1,4 - 二氢吡啶（DHP）钙拮抗剂结合位点的亲和力（Kd）、密度（Bmax）和选择性的影响。相对于需氧对照，30分钟和60分钟的常温（37℃）缺氧均未影响这些位点的Bmax或Kd。60分钟（而非30分钟）缺氧后的15分钟复氧降低了DHP结合位点的密度，而未改变其亲和力或选择性。在37℃下用0.1 mM DNP（氧化磷酸化解偶联剂）进行需氧灌注30分钟，无论有无葡萄糖存在，Bmax均增加（P＜0.05），分别增加48%和27%，而Kd无变化。在进一步用DNP灌注30分钟期间，Bmax的这种增加减弱。用1.0 mM IAA和0.1 mM DNP灌注30分钟导致DHP结合位点的Bmax显著增加（27%）。再用IAA和DNP灌注30分钟使Bmax恢复到对照水平。在此条件下Kd未改变。无论灌注条件如何，结合位点的选择性不变，在置换结合的（+）[³H]PN200 - 110时，（+）PN200 - 100＞（ - ）Bay K8644＞（ - ）PN200 - 110 =（+）Bay K8644。在所有条件下，（ - ）D600总是与DHP结合位点发生变构相互作用，并且结合受到d - 顺式地尔硫䓬的刺激。这些结果表明，缺氧和化学诱导的ATP耗竭均不能模拟缺血对心脏DHP结合位点的影响。
