Sodium Pump Inhibitors In Blood Pressure Regulation

研究表明，两种内源性钠泵配体（SPL），内源性哇巴因（EO）和海蟾毒配基（MBG），在哺乳动物组织中共存。MBG作为哇巴因抗性Na/K-ATP酶（NKA）α-1亚型的选择性抑制剂，NKA是肾脏、血管平滑肌和成人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NaCl敏感性高血压发展的基础，脑EO触发外周MBG，从而升高血压。体内给药MBG抗体高血压DS降低血压。在过去的一年中，我们的研究集中在：（i）在Dahl高血压建立过程中中心和外周SPL的关系的研究，（ii）SPL和蛋白激酶在肾和心血管NKA中的相互作用的研究，和（iii）MBG在先兆子痫中的致病作用的研究。 (i)NaCl敏感性高血压的发病机制 在DS中NaCl高血压的发展过程中，海马和杏仁核中EO的初始瞬时升高，随后下丘脑和垂体视上核中EO的增加，刺激垂体血管紧张素II（ATII），其通过ATII敏感通路刺激肾上腺皮质产生MBG。发生MBG产生的持续增加，并诱导肾小管和心血管组织中钠泵的抑制。后者有助于慢性血压升高引起的持续高氯化钠摄入量。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模拟NaCl负荷的作用，并导致MBG水平增加，以及利钠和升压反应。通过体内施用抗MBG抗体来防止中枢哇巴因施用的这些作用。因此，两种SPL，MBG和EO，在这个恶性循环中起着重要作用，这可能是NaCl敏感性高血压发病机制的基础。 (ii)蛋白激酶调节MBG诱导的NKA抑制。 蛋白激酶使NKA磷酸化/去磷酸化并改变其对洋地黄样配体的敏感性。在大鼠主动脉中，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，在肾髓质中，ANP诱导α-1 NKA磷酸化并增强MBG诱导的钠泵抑制。ANP的这些作用由蛋白激酶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PKG的2型和1型亚型，因此这两种亚型可能对NKA对MBG的敏感性表现出相反的作用。血管舒张剂ANP和血管收缩剂MBG的同时产生，其发生在NaCl敏感性高血压和充血性心力衰竭中，导致其利钠作用的增强，而ANP肽可以抵消MBG的有害血管收缩作用。因此，MBG的促高血压心血管效应可能通过cGMP/PKG依赖性机制被拮抗。在高血压Dahl大鼠中，心脏NKA对MBG的敏感性增强与心肌中β 2和δ PKC的上调有关。用抗高血压化合物西来他宁长期治疗Dahl大鼠，其抑制PKC，降低血压，使心脏NKA对MBG脱敏并降低其磷酸化，并防止心肌肌膜中PKC的增加。因此，SPL和蛋白激酶对心血管NKA的相互作用是治疗高血压的新靶点。 (iii)先兆子痫。 以前我们已经表明，血浆MBG的显着增加伴随先兆子痫。有趣的是，地高辛抗体（DIGIBIND）由于能够结合SPL，因此在临床上用于治疗先兆子痫。在两种先兆子痫大鼠模型中（妊娠期补充NaCl，给予和不给予醋酸脱氧皮质酮- DOCA），血压升高与MBG水平持续升高相关，但与EO无关。此外，妊娠大鼠的DOCA+NaCl处理与子宫脉管系统对MBG收缩作用的敏感性显著增加相关。给妊娠高血压大鼠施用MBG的多克隆和单克隆抗体可降低血压并恢复血管NKA的活性。这些数据使MBG成为先兆子痫患者治疗的潜在靶点。 总之，这些发现表明MBG是高血压发病机制中的一个新因素，并可能在高血压治疗中开辟新的药理学可能性，包括在先兆子痫中用特异性抗体阻断循环MBG，通过抑制蛋白激酶C减弱MBG对心血管系统NKA的NKA抑制作用，并导致钠泵对MBG的脱敏，以及通过ACE抑制剂和/或血管紧张素II受体阻断剂抑制肾上腺皮质MBG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.