摘要目的：研究羟苯氨酮(oxyphenamone, Oxy)强心作用的生化机理。方法：采用Na⁺,K⁺-ATP酶活性和cAMP-PDE活性、肌浆网Ca²⁺-ATP酶活性和cAMP含量以及心肌肌原纤维Ca²⁺,Mg²⁺-ATP酶活性等测定法，研究Oxy对它们的影响，并与milrinone和MCI-154作比较。结果：Oxy对Na⁺,K⁺-ATP酶和PDE无抑制作用，也不影响心肌cAMP含量，但能显著增强心肌肌原纤维对Ca²⁺的敏感性，高浓度时轻度抑制心肌肌浆网Ca²⁺-ATP酶活性。结论：Oxy的强心作用方式不同于强心苷、β受体激动剂和PDE抑制剂等强心药,可能为一种新的钙增敏性强心药物。

BIOCHEMCAL MECHANISM OF THE POSITIVE

INOTROPIC EFFECT OF OXYPHENAMONE

Ye Yixin(Ye YX),Yang Xiaoran(Yang XR),

Qiao Xiaoying(Qiao XY) and Chen Lanlan(Chen LL)

(Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and

Peking Union Medial College, Beijing 100005)

Fan Lili(Fan LL) Lin Yong(Lin Y)

(Institute of Meteria Medica Chinese Academy of Medical Sciences and Peking Union Medial College, Beijing 100050)

ABSTRACTAIM: To investigated the biochemical mechanisms of the positive inotropic effect of oxyphenamone(Oxy).METHODS: The assays of Na⁺,K⁺-ATPase activity, cAMP dependent phosphodiesterase(cAMP-PDE) activity and Ca²⁺-ATPase activity in sarcoplasmic reticulum(SR) isolated from cardiac muscle, cAMP level in cardiac muscle and the cardiac myofibrillar Ca²⁺,Mg²⁺-ATPase activity were adopted and compared with those of strophanthin-G（Str） and milrinone（Mil）.RESULTS: Oxy at its effective concentration, showed no remarkable inhibition on Na⁺,K⁺-ATPase and cAMP dependent phosphodiesterase (cAMP-PDE) activities, while in parallel experiments Na⁺,K⁺-ATPase and cAMP-PDE activities were significantly inhibited by Str and Mil. Their IC₅₀ values were found to be 2.0 μmol^.L^-1, and 85 μmol^.L^-1, respectively. Oxy did not affect the cAMP level in cardiac muscle of guinea pig. However, Mil at 30 μmol^.L^-1 in control experiments increased the cAMP level by 73.6%. These results suggest that the mechanism of the positive inotropic effect of Oxy differs from that of glycosides, PDE inhibitors and β-adrenergic agonists. Oxy at 100 μmol^.L^-1 inhibited Ca²⁺-ATPase activity significantly in cardiac sarcoplasmic reticulum. Its IC₅₀ value was 200 μmol^.L^-1. The result suggests that Oxy at high concentration exerts inhibitory effect on the Ca²⁺ uptake by SR. This mechanism may be partly responsible for the positive inotropic effect of Oxy. Oxy at 50 μmol^.L^-1 shifted the relationship curve between pCa²⁺and myofibrillar Ca²⁺,Mg²⁺-ATPase activity to the left without affecting the maximum enzyme activity. When pCa 7, Oxy increased the myofibrillar Ca²⁺,Mg²⁺-ATPase activity in a concentration dependent manner and EC₅₀ value was about 10 μmol^.L^-1. MCI-154 at 100 μmol^.L^-1 and some new derivatives of Oxy with positive inotropic effect enhanced the Ca²⁺ sensitivity. Mil at 100 μmol^.L^-1and some new derivatives of Oxy with no positive inotropic effect showed no effect at all. Solaro and Kitada found a positive correlation between the increase of myofibrillar Ca²⁺,Mg²⁺-ATPase activity and the enhancement of Ca²⁺ sensitivity of the contractile protein system.CONCLUSION: These results demonstrate that the biochemical mechanism of the positive inotropic effect of Oxy is different from these of the cardiac glycosides, PDE inhibitors and β-adrenergic agonists, therefore, it may be a novel cardiotonic agent, a calcium sensitizer.

KEY WORDSoxyphenamone; Na⁺,K⁺-ATPase; phosphodiesterase; Ca²⁺,Mg²⁺-ATPase; cAMP; calcium sensitizers

羟苯氨酮(oxyphenamone, Oxy)系中国医学科学院药物研究所合成的新化合物，经离体、整体和分子水平的实验研究表明有显著的强心和扩血管作用^［1,2］。为探讨Oxy强心作用的生化机理，本文研究了Oxy对Na⁺,K⁺-ATP酶、磷酸二酯酶(PDE)、cAMP含量和肌浆网Ca²⁺-ATP酶以及去膜心肌肌原纤维钙敏性的影响。

材料和方法

材料羟苯氨酮由我院药物研究所合成室提供,用蒸馏水稍加温制成母液。 米利农(milrinone, Mil)由重庆医工所提供,用少量0.1 mol^.L^-1 HCl溶解后用碱液调至pH中性备用。毒毛旋花子甙G(strophanthin-G, Str)购自Sigma公司,用50%～60%乙醇溶解后用缓冲液稀释配成母液,乙醇的终浓度不超过1%。 上述母液的浓度均为10 mmol^.L^-1，实验前用缓冲液稀释至所需浓度。 ³H-cAMP 购自中国原子能研究所,比活性为1028.6 GBq^.mmol^-1。

Na⁺,K⁺-ATP酶制备及其活性测定^［3］用经典方法从猫肾皮质提取纯化得到较纯的Na⁺,K⁺-ATP酶制备,制备的比活性为30～40 u^.mg^-1(蛋白)，占总酶的(90～95)％，浓度为1 mg(蛋白)^.ml^-1。测定酶活性的方法简述如下: 反应液成分(mmol^.L^-1): NaCl 100, MgCl₂ 5, KCl 15, EDTA 0.1 和Tris-HCl 50, pH 7.4。 酶制备约0.1 mg^.ml^-1, 总体积1 ml。 空白管不加酶制备,Mg²⁺-ATP酶管加Str 1 mmol^.L^-1，总酶管不加Str, 药物管加不同浓度测试药物。 37℃保温10 min后,加入Na₂ATP 4 mmol^.L^-1启动反应。10 min后,加15%三氯醋酸(TCA)终止反应。 各管置冰浴降温10 min,离心(3 000 r^.min^-1,常温) 10 min取上清液1 ml, 按Bonting法^［4］测释放的无机磷量。酶活性单位为μmolPi^.mg^-1(蛋白)^.h^-1，总酶和Mg²⁺-ATP酶的差即为Na⁺,K⁺-ATP酶活性。药物对酶活性的影响按下式计算: %对照值=加药管酶活性/无药管酶活性×100。

磷酸二酯酶(PDE)的制备及其活性测定参考文献^［5］法,取豚鼠心室肌约1 g加5倍量缓冲液(mmol^.L^-1: MgCl₂ 5, Tris-HCl 50),离心(8 000×g, 4℃) 30 min, 上清液为酶制备,置-20℃冰箱保存,用前加缓冲液稀释。

cAMP-PDE(cGAP-PDE)活性测定原理:

用QAE-Sephadex柱分离 ［³H］cAMP(cGMP)和［³H］腺苷,测定［³H］腺苷的放射性,以［³H］cAMP转化成［³H］腺苷的转化率表示PDE活性。药物对酶活性的影响的计算同Na⁺,K⁺-ATP酶。测定PDE酶的方法简述如下: 反应液的成分(mmol^.L^-1): MgCl₂ 5, Tris-HCl 50, pH 7.4, ［³H］cAMP(cGMP)cpm为80 000～100 000 min^-1, 一定量酶制备和测试药物,总体积250 μl。 空白管加入灭活酶制备或不加酶制备,37℃反应15 min后,置沸水75 s终止反应。 冷却后加入蛇毒(10mg^.ml^-1) 15 μl,再温育10 min(37℃),加腺苷(25 mmol^.L^-1) 735 μl，然后将全部液体倒入色谱柱,用10 ml小瓶收集。待液体全部流出后,加甲酸铵(20 mmol^.L^-1)4 ml洗脱。<