Objective To evaluate the role of endogenous ouabain(EO)In the development of hypertension and the characteristics of EO secretion in 1k1c(one kidney, one clipped) hypertensive rats. Methods EO content of serum and tissues in 1k1c hypertensive rats and normal control Sprague-Dawley (SD) rats was detected by the method of enzyme linked immunosorbent assay (ELISA). The relationship between serum or tissue ouabain and blood pressure was analyzed in 1k1c hypertensive rats. Results The ouabain content of serum, heart, kidney, adrenal gland, pituitary and hypothalamus was significantly higher in 1k1c hypertensive rats then that in normal control SD rats (2.25, 2.63, 3.35,40.37,3.34,15.7μg/kg tissue in 1k1c hypertensive rats vs 1.12, 1.79, 1.73, 27.54, 1.83, 10.10μg/kg tissue in control SD rats, respectively. P<0.05 for all of these comparisons). The ouabain content of the adrenal gland and the hypothalamus was higher than that of other tissues or serum, both in 1k1c rats and in control SD rats. The EO content of serum, kidney and hypothalamus was significantly correlated with blood pressure in 1k1c hypertensive rats (r=0.59,0.63,0.52, respectively. P<0.05). The ouabain content of heart, liver, adrenal gland and pituitary was not correlated with blood pressure. Conclusions EO might play an important role in the development of hypertension in 1k1c hypertensive rats. The adrenal gland may be a major source of EO and the hypothalamus-pituitary-adrenal axis may be involved in the regulation of EO secretion.

This study was designed to evaluate the role of endogenous ouabain (EO) in the development of hypertension in 1k1c (one kidney, one clip) hypertensive rats. First, the EO content of the serum of 1k1c hypertensive rats and normal Sprague-Dawley (SD) rats was detected by the enzyme linked immunosorbent assay method (ELISA). Second, blood pressure changes in the 1k1c rats were recorded directly after the 1k1c rats were injected randomly with anti-ouabain antibody, normal rabbit lgG, and normal saline, respectively. The results showed that EO levels in the serum of 1k1c hypertensive rats were significantly higher than those of normal SD rats (2.25± 0.92μg/l, P<0.01), and correlated significantly with systolic blood pressure (r=0.59, P<0.05). Anti-ouabain antibody was able to significantly decrease the blood pressure of 1k1c hypertensive rats in a dose-dependent manner, while normal rabbit lgG or normal saline was not. These results indicate that endogenous ouabain might play an important role in the development of hypertension in 1k1c hypertensive rats.

The effects of ouabain and digoxin on both the systolic blood pressure (SBP) and sodium pumpα-subunit expression in some tissues of rats were compared. Normal rats were injected with ouabain, digoxin, and normal saline (NS), respectively, everyday, and indirect SBP was recorded once a week. Six weeks later, all the rats were killed, and sodium pump α1-, α2-, and α3-subunit mRNA levels were detected in the myocardium, kidney, adrenal gland, aortic smooth muscle, and hypothalamus by the RT-PCR method. The results showed that the SBP of rats infused with ouabain increased significantly at the end of week 6, while no difference in SBP was found between the digoxin and NS groups. The effects of ouabain and digoxin on sodium pump α-subunit isoform expression were also different. Myocardium: both ouabain and digoxin stimulated expression of the α3-isoform whereas α2 was unchanged. Levels of the α1 isoform decreased significantly in the ouabain group and decreased slightly in the digoxin group, respectively. Kidney: digoxin had the same effects as ouabain. α1 levels increased, but those of α2 and α3 remained unchanged. Adrenal gland: α2 and α3 levels increased, but those of α1 decreased in the ouabain group. α1 and α3 levels increased and those of α2 remained unchanged in the digoxin group. Aortic smooth muscle: both ouabain and digoxin increased α1 and α3 expression. α2 levels decreased in the digoxin group but remained unchanged in the ouabain group. Hypothalamus: both ouabain and digoxin stimulated α1 expression, while α2 and α3 levels remained unchanged. The results of this study have shown that ouabain and digoxin have the different effects on both the systolic blood pressure and expression of sodium pump α-subunit isoforms in some tissues in rats. Further studies on the expression of sodium pump α-subunit isoforms might be helpful for the understanding of the physilolgical role endogenous ouabain and the molecular mechanisms involved in the pathogenesis of hypertension.

目的 对比研究哇巴因与地高辛对大鼠垂体钠泵（Na+，K+-ATP 酶）α亚单位基因表达的影响，探讨内源性哇巴因（EO）的生物学效应以及洋地黄类药物理作用的分子机制。方法 长期给予大鼠注射小剂量哇巴因（20μg·kg-1·d-1）与地高辛（32μg·kg-1·d-1），动态观察大鼠血压变化；分别应用分子生物学RT-PCR 及免疫组织化学技术，探讨各组大鼠垂体钠泵α1、α2 及α3 亚单位mRNA 及蛋白水平基因表达的改变。结果 哇巴因与地高辛对大鼠血压的影响存在着明显差别，长期给予大鼠注射小剂量哇巴因可使大鼠血压升颃是地高辛对大鼠血压无影响。哇巴因与地高辛对垂体钠泵α亚单位表达的影响存在明显大同：在mRNA 水平，哇巴因使α1、α2 及α3 亚单位的表在均明显减弱，而地高辛仅使α3 亚单位表达减弱；在蛋白水平，哇巴因使α2 及α3 亚单位表达减弱，α1 亚单位无改变，而地高辛使α1 及α3 亚单位表达减弱，α2 亚单位无改变。结论 哇巴因在高血压发病中可能起着重要作用；哇巴因与地高辛可导致不同的钠泵基因表达改变，为进一步提示EO 生理与病理作用以及洋地黄类药物药理与毒理作用的分子机制提供了理论及实验依据。

目的 制备哇巴因多克隆抗体F（ab）2 片段，并对其进行分子量的测定。方法 免疫家兔制备哇巴因多克隆抗体，在适宜的条件下，用胃蛋白酶酶解哇巴因多克隆抗体，制备F（ab）2 片段。酶解产物经HPSEC 分析、纯化，ELISA法检测其免疫学活性。并在适宜的色谱条件下，分别测定三种标准蛋白的洗脱体积，绘制标准曲线，测定其分子。结果100mg哇巴因多克隆抗体在2mg 胃蛋白酶消化18h，反应体系pH3.0的条件下消化，制备出了活性的F（ab）2 片段，其相对分子质量为107kD。结论 用胃蛋白酶消化哇巴因多克隆抗体，可制备出活性的F（ab）2片段，同时其相对分子质量的测定为其进一步研究提供了依据。