Schmidt T A, Svendsen J H, Haunsø S, Kjeldsen K
Department of Medicine B, Rigshospitalet, University of Copenhagen School of Medicine, Denmark.
Basic Res Cardiol. 1990 Jul-Aug;85(4):411-27. doi: 10.1007/BF01907133.
Na,K-ATPase concentration was measured by vanadate facilitated 3H-ouabain binding to intact samples taken from various parts of porcine and canine myocardium. In porcine and canine heart 3H-ouabain binding site concentration in ventricles was 1.4-2.5 times larger than in atria. Evaluation of 3H-ouabain binding kinetics revealed no major difference between atria and ventricles: Equilibrium was obtained after the same incubation time in right atrium (RA) as in left ventricle (LV), both in porcine and canine heart. Unspecific uptake and retention of 3H-ouabain was for porcine heart RA and LV 1.5 and 1.4, respectively, and for canine heart RA and LV, both 1.2% filling (i.e., volume (ml) of incubation medium 3H-radioactivity taken up per mass unit (g wet wt.) of tissue multiplied by 100). The apparent dissociation constant (KD) was 1.4 x 10(-8) and 1.9 x 10(-8) in porcine RA and LV and 2.6 x 10(-8) and 6.1 x 10(-8) mol/l in canine RA and LV. Loss of specifically bound 3H-ouabain during the washout procedure occurred with a half-life time (T1/2) of 16.7 and 28.6 in RA and LV of porcine heart and 91.2 and 151.6 h in RA and LV of canine heart. Duly corrected for these errors of the method--factor 1.16 and 1.13, respectively, for porcine RA and LV, and factor 1.11 and 1.13 for canine RA and LV, total 3H-ouabain binding site concentration was found to be 553 +/- 74 and 1037 +/- 45 pmol/g wet wt. (means +/- SEM, n = 5) in porcine RA and LV, and 569 +/- 37 and 1410 +/- 40 pmol/g wet wt. (means +/- SEM, n = 5) in the canine RA and LV. These values were confirmed by measurements of 3H-digoxin binding to the porcine heart. The present quantification of myocardial Na,K-ATPase gives values up to 154 times higher than measurements based upon Na,K-ATPase activities in membrane fractions where the recovery of Na,K-ATPase may be less than 1% due to loss during purification. A higher Na,K-ATPase concentration is found in small animals than in large animals. A relationship between higher concentration of Na,K-ATPase and larger pressure work in ventricles compared to atria is suggested. Myocardial 3H-ouabain binding sites were found to be stable for 20 min of ischemia, followed by 1 h of reperfusion, supporting the concept that myocyte injury induced by short term ischemia may be reversible and that reperfusion may result in normalization.
通过钒酸盐促进3H-哇巴因与取自猪和犬心肌不同部位的完整样本结合来测量钠钾ATP酶（Na,K-ATPase）的浓度。在猪和犬的心脏中，心室的3H-哇巴因结合位点浓度比心房大1.4至2.5倍。对3H-哇巴因结合动力学的评估显示心房和心室之间没有重大差异：在猪和犬的心脏中，右心房（RA）和左心室（LV）在相同的孵育时间后达到平衡。猪心脏RA和LV中3H-哇巴因的非特异性摄取和保留分别为1.5%和1.4%，犬心脏RA和LV均为1.2%填充（即每克湿重组织摄取的孵育培养基3H放射性的体积（毫升）乘以100）。猪RA和LV中的表观解离常数（KD）分别为1.4×10⁻⁸和1.9×10⁻⁸，犬RA和LV中为2.6×10⁻⁸和6.1×10⁻⁸摩尔/升。在洗脱过程中，猪心脏RA和LV中特异性结合的3H-哇巴因的半衰期（T1/2）分别为16.7小时和28.6小时，犬心脏RA和LV中为91.2小时和151.6小时。对该方法的这些误差进行适当校正后——猪RA和LV分别为1.16和1.13因子，犬RA和LV分别为1.11和1.13因子，发现猪RA和LV中总3H-哇巴因结合位点浓度分别为553±74和1037±45皮摩尔/克湿重（平均值±标准误，n = 5），犬RA和LV中为569±37和1410±40皮摩尔/克湿重（平均值±标准误，n = 5）。这些值通过测量3H-地高辛与猪心脏的结合得到了证实。目前对心肌钠钾ATP酶的定量结果比基于膜分离物中钠钾ATP酶活性的测量值高出多达154倍，在膜分离物中，由于纯化过程中的损失，钠钾ATP酶的回收率可能低于1%。在小动物中发现的钠钾ATP酶浓度高于大动物。提示心室中钠钾ATP酶浓度较高与比心房更大的压力功之间存在关系。发现心肌3H-哇巴因结合位点在缺血20分钟后再灌注1小时内是稳定的，这支持了短期缺血诱导的心肌细胞损伤可能是可逆的且再灌注可能导致恢复正常的概念。
