Sodium Pump Inhibitors In Blood Pressure Regulation

以往的研究表明，哺乳动物组织中同时存在两种内源性钠泵配体(SPL)：内源性哇巴因(EO)和华蟾素(MBG)。MBG是哇巴因耐药的Na/K-ATPase(NKA)α-1亚型的选择性抑制剂，NKA是肾脏、血管平滑肌和成年心肌细胞中的主要钠泵亚型。在Dahl盐敏感大鼠(DS)中，α-1 NKA的缺陷是盐敏感性高血压发生的基础，脑内EO触发外周MBG，从而使血压升高。体内注射抗高血压DS的MBG抗体可降低血压。在过去的一年里，我们的研究集中在：(I)Dahl高血压形成过程中中枢和外周SPL的关系的研究，(Ii)SPL和蛋白激酶对肾脏和心血管NKA的相互作用的研究，以及(Iii)MBG在子痫前期发病中的作用的研究。 (I)盐敏感型高血压的发病机制。 在DS的氯化钠高血压的发展过程中，海马区和杏仁体的EO开始短暂升高，随后下丘脑视上核和垂体的EO增加，刺激垂体血管紧张素II(ATII)，从而通过ATII敏感的途径刺激肾上腺皮质产生MBG。MBG的产量持续增加，并导致肾小管和心血管组织中的钠泵受到抑制。后者导致持续高盐摄入量引起的慢性血压升高。MBG抑制肾小管钠转运的复杂机制涉及NKA的内化。双侧给予低浓度哇巴因可模拟氯化钠负荷的影响，并导致MBG水平升高，以及利钠和升压反应。中枢哇巴因的这些作用可通过体内注射抗MBG抗体来预防。因此，两种SPL，MBG和EO，在这种恶性循环中起着重要作用，这可能是盐敏感型高血压发病的基础。 (Ii)蛋白激酶对MBG诱导的NKA抑制有调节作用。 蛋白激酶使NKA磷酸化/去磷酸化，并改变其对洋地黄类配体的敏感性。在大鼠主动脉，纳摩尔浓度的ANP使α-1 NKA去磷酸化，并显著降低钠泵对MBG的敏感性。相反，在肾髓质，ANP诱导α-1 NKA磷酸化，并加强MBG对钠泵的抑制。ANP的这些作用可被蛋白激酶G的激活剂8-BR-PET-cGMP模拟。由于肾脏和血管平滑肌细胞分别表达2型和1型PKG亚型，这两种亚型可能对NKA对MBG的敏感性产生相反的影响。在盐敏感型高血压和充血性心力衰竭中，同时产生血管舒张剂ANP和血管收缩药MBG，导致它们的利钠作用增强，而ANP多肽可能抵消MBG的有害血管收缩作用。因此，MBG的高血压心血管效应可能是通过cGMP/PKG依赖机制被拮抗的。在高血压Dahl大鼠中，心脏NKA对MBG的敏感性增强与心肌中Beta2和Delta PKC的上调有关。对Dahl大鼠长期应用抗高血压化合物cicletanine治疗，可抑制PKC，降低血压，使心脏NKA对MBG脱敏，减少其磷酸化，并防止心肌肌膜PKC增加。因此，SPL和蛋白激酶对心血管NKA的相互作用是高血压治疗的新靶点。 (Iii)先兆子痫。 以前我们已经证明，血浆MBG的急剧增加伴随着子痫前期。有趣的是，地高辛的抗体(DigiBind)，由于其结合SPL的能力，在临床上被用于治疗先兆子痫。在两种先兆子痫大鼠模型(孕妇补充氯化钠并服用和不服用醋酸脱氧皮质酮-DOCA)中，血压的升高与MBG水平的持续升高有关，但与EO水平的持续升高无关。此外，DOCA+氯化钠处理妊娠大鼠与子宫血管对MBG收缩作用的敏感性显著增加有关。孕期高血压大鼠应用抗MBG多克隆和单抗可降低血压，恢复血管NKA活性。这些数据使MBG成为先兆子痫患者治疗的潜在靶点。 综上所述，这些发现表明MBG是高血压发病机制中的一个新的因子，并可能为高血压的治疗开辟新的药理学可能性，包括在子痫前期用特异性抗体阻断循环中的MBG，通过抑制蛋白激酶C而减弱MBG对心血管NKA的抑制作用而导致钠泵对MBG的脱敏，以及通过ACE抑制剂和/或血管紧张素II受体阻滞剂抑制肾上腺皮质MBG的产生。

Previous studies demonstrated that two endogenous sodium pump ligands (SPL), endogenous ouabain (EO) and marinobufagenin (MBG), coexist in mammalian tissues. MBG acts as a selective inhibitor of ouabain resistant alpha-1 isoform of Na/K-ATPase (NKA), the main sodium pump isoform in the kidney, vascular smooth muscle and adult myocardiocytes. In Dahl salt-sensitive rats (DS), in which the defect of alpha-1 NKA underlies development of NaCl sensitive hypertension, brain EO triggers peripheral MBG, which raises the blood pressure. In vivo administration of MBG antibody to hypertensive DS lowered the blood pressure. During the past year, our research focused on: (i) studies of relationship between central and peripheral SPL during establishment of Dahl hypertension, (ii) studies of interaction of SPL and protein kinases on renal and cardiovascular NKA, and (iii) investigations of the pathogenic role of MBG in preeclampsia. (i) Pathogenesis of NaCl sensitive hypertension. In the course of development of NaCl hypertension in DS, an initial transient rise of EO in the hippocampus and amygdala, followed by an increase in EO in the supraoptic nucleus of the hypothalamus and pituitary, stimulates pituitary angiotensin II (ATII), which stimulates adrenocortical production of MBG via an ATII sensitive pathway. A sustained increase in MBG production occurs, and induces inhibition of the sodium pump in renal tubules and in cardiovascular tissues. The latter contributes to a chronic blood pressure elevation induced by sustained high NaCl intake. The complex mechanism by which MBG inhibits sodium transport in renal tubules involves internalization of the NKA. Bilateral administration of low concentration of ouabain mimics the effects of NaCl loading and results in increased levels of MBG, and natriuretic and pressor responses. These effects of central ouabain administration are prevented by in vivo administration of anti-MBG antibody. Thus, two SPL, MBG and EO, play an important role in this vicious circle, which may underlie the pathogenesis of NaCl sensitive hypertension. (ii) Protein kinases modulate MBG induced NKA inhibition. Protein kinases phosphorylate/dephosphorylate the NKA and alter its sensitivity to digitalis-like ligands. In rat aorta, ANP at nanomolar concentration dephosphorylates alpha-1 NKA and markedly reduces the sensitivity of the sodium pump to MBG. In contrast, in renal medulla, ANP induces alpha-1 NKA phosphorylation and potentiates MBG induced inhibition of the sodium pump. These effects of ANP are mimicked by an activator of protein kinase G, 8-Br-PET-cGMP. Since kidney and vascular smooth muscle cells express type 2 and type 1 isoforms of the PKG, respectively, these two isoforms are likely to exhibit opposite effects on the sensitivity of NKA to MBG. The concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, MBG, which occurs in NaCl sensitive hypertension and in congestive heart failure, results in the potentiation of their natriuretic effects, while ANP peptides may offset the deleterious vasoconstrictor effect of MBG. Therefore, prohypertensive cardiovascular effects of MBG may be antagonized via cGMP/PKG dependent mechanisms. In hypertensive Dahl rats, enhanced sensitivity of cardiac NKA to MBG is associated with upregulation of beta2 and delta PKC in the myocardium. Chronic treatment of Dahl rats with cicletanine, an antihypertensive compound, which inhibits the PKC, lowers the blood pressure, desensitizes cardiac NKA to MBG and decreases its phosphorylation, and prevents increases in PKC in myocardial sarcolemma. Thus, interaction of SPL and protein kinases on cardiovascular NKA is a novel target for therapy of hypertension. (iii) Preeclampsia. Previously we have shown that dramatic increases in plasma MBG accompany preeclampsia. Interestingly, antibodies to digoxin (DIGIBIND), due to their ability to bind SPL are used in the treatment of preeclampsia clinically. In two rat models of preeclampsia (NaCl supplementation of pregnant with and without administration of deoxycorticosterone acetate- DOCA) elevations of blood pressure are associated with sustained increases in the levels of MBG, but not EO. Furthermore, DOCA+NaCl treatment of pregnant rats is associated with a dramatic increase in the sensitivity of uterine vasculature to constrictor effect of MBG. Administration of polyclonal and monoclonal antibodies to MBG to pregnant hypertensive rats lowers the blood pressure and restores the activity of vascular NKA. These data make MBG a potential target for therapy in patients with preeclampsia. Taken together, these findings demonstrate that MBG is a novel factor in the pathogenesis of hypertension, and may open new pharmacological possibilities in the treatment of hypertension, including blockade of circulating MBG with specific antibodies in preeclampsia, attenuation of NKA inhibitory effect of MBG on cardiovascualar NKA by inhibition of protein kinase C and resulting desensitization of the sodium pump to MBG, and inhibition of adrenocortical MBG production by ACE inhibitors and/or angiotensin II receptor blockers.