Sodium Pump Inhibitors In Blood Pressure Regulation

两种内源性洋地黄样强心类固醇（CTS），内源性哇巴因（EO）和海蟾毒配基（MBG），共存于哺乳动物组织中。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（VSM）和成人心肌细胞中的主要亚型。在Dahl盐敏感大鼠（DS）中，摄入高NaCl，脑EO触发外周MBG，从而升高血压（BP）。在先兆子痫（PE）的大鼠模型中，体内施用单克隆抗体（Mab）降低BP。在过去的一年里，我们的研究集中在（i）中央和外周CTS之间的关系在DS，（ii）治疗性抗MBG单克隆抗体（Mab）的发展，和（iii）MBG诱导的NKA介导的细胞信号的衰老相关机制的研究。 (i)NaCl敏感性高血压的发病机制 DS的NaCl负荷后，海马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（AngII），并通过激活交感神经系统激活肾上腺皮质肾素-血管紧张素系统（RAS）。肾上腺皮质AngII通过AT 1受体作用刺激MBG的产生。MBG产生的增加诱导肾小管和VSM中Na泵的抑制。在过去的一年中，我们重新分析了NaCl诱导的哇巴因介导的促高血压信号的事件序列。我们研究了急性氯化钠负荷和低剂量哇巴因中枢给药后，大鼠海马和下丘脑视上核内注射抗哇巴因抗体的影响。用特异性抗体阻断脑EO可防止（a）刺激MBG产生，（B）血压升高，和（c）由中枢给药哇巴因和NaCl负荷诱导的尿钠排泄和肾上腺皮质RAS激活。 (ii)治疗性单克隆抗MBG抗体的开发。 在PE患者和实验室大鼠、高血压受试者和终末期肾病（ESRD）大鼠以及高血压DS中，MBG水平升高与BP升高相关。在过去的一年中，我们证明了3E 9 anti-MBG Mab有效地降低了患有ESRD的高血压大鼠和Ang II诱导的高血压大鼠的血压。3E 9 Mab还离体逆转了从PE患者获得的红细胞中钠泵的抑制。3E 9 Mab的人源化正在进行中。 (iii)衰老，MBG诱导的细胞信号传导及其调节机制。 心房利钠肽（ANP）通过cGMP依赖性机制使血管平滑肌（VSM）NKA脱磷酸化，并显著降低其对MBG的敏感性。在大鼠肾髓质中，ANP表现出相反的作用，即，诱导NKA磷酸化并使Na泵对MBG的抑制作用敏感。由于VSM和肾髓质分别表达PKG 1和PKG 2亚型，这两种PKG亚型可能介导ANP对NKA磷酸化和MBG敏感性的相反作用。因此，血管舒张剂ANP和血管收缩剂MBG的同时产生加强彼此的利钠作用，但ANP可能抵消MBG的血管收缩作用。衰老与CTS水平升高以及肾脏和VSM中cGMP-PKG信号传导下降相关。这种下降可能使MBG和ANP对肾和心血管NKA的作用之间的平衡向肾Na泵抑制较小和VSM NKA抑制较大的方向移动，从而有助于高血压的发病机制。在老年SD大鼠（S-D），急性NaCl负荷引起的升压反应和MBG反应较青年大鼠明显增强，而利钠反应较青年大鼠明显减弱。我们还比较了ANP对MBG引起的青年和老年大鼠VSM和肾髓质NKA抑制的影响。在青年大鼠中，ANP可增强MBG对肾NKA的抑制作用，并逆转VSM对NKA的抑制作用;而在老年大鼠中，ANP的作用则相反：ANP不改变MBG对肾NKA的抑制作用，而在VSM中，ANP可增强MBG对NKA的抑制作用。因此，在老年大鼠中，ANP不能增强MBG的生理作用，尿钠排泄，同样，衰老与ANP抵消MBG诱导的血管收缩的能力的丧失有关。盐敏感性高血压的表型在许多方面类似于衰老期间发生的表型。在我们的实验中，NaCl负荷的盐敏感性DS和血压正常的S-D，在存在可比的MBG反应，产生优先抑制钠泵在血管平滑肌的DS和肾脏的S-D。因此，NaCl负荷的DS表现出升压反应和钠潴留，而在S-D中，在没有BP升高的情况下发生足够的钠尿排泄。因此，在大鼠中，BP的盐敏感性与肾Na泵对MBG的反应性丧失相关，而血管Na泵表现出更高的MBG敏感性。

Two endogenous digitalis-like cardiotonic steroids (CTS), 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 isoform in the kidney, vascular smooth muscle (VSM) and adult cardiomyocytes. In Dahl salt-sensitive rats (DS) on a high NaCl intake, brain EO triggers peripheral MBG, which raises the blood pressure (BP). In a rat model of preeclampsia (PE), the in vivo, administration of monoclonal antibody (Mab) lowers the BP. During the past year our research focused on (i) the relationship between central and peripheral CTS in DS, (ii) the development of a therapeutic anti-MBG monoclonal antibody (Mab), and (iii) studies of aging-related mechanisms of MBG-induced NKA-mediated cell signaling. (i) Pathogenesis of NaCl-sensitive hypertension. Following NaCl loading of DS, an initial transient rise of hippocampal EO, followed by an increase in EO in the supraoptic nucleus of the hypothalamus and pituitary, stimulates pituitary angiotensin II (AngII), and, via activation of sympathetic nervous system, activates the adrenocortical renin-angiotensin system (RAS). Adrenocortical AngII acting through AT1 receptors stimulates production of MBG. An increase in MBG production induces inhibition of the Na pump in renal tubules and in VSM. During the past year, we pharmacologically analyzed the sequence of events underlying NaCl-induced ouabain-mediated prohypertensive signaling. We studied effects of administration of anti-ouabain antibodies into hippocampus and supraoptical nucleus of hypothalamus in rats following acute NaCl loading and central administration of low doses of ouabain. Blockade of brain EO with a specific antibody prevented (a) stimulation of MBG production, (b) elevation of BP, and (c) natriuresis and activation of adrenocortical RAS induced by both centrally-administered ouabain and NaCl loading. (ii) Development of a therapeutic monoclonal anti-MBG antibody. Heightened levels of MBG are associated with elevations of BP in patients and laboratory rats with PE, in hypertensive subjects and in rats with end stage renal disease (ESRD), and in hypertensive DS. During the past year, we demonstrated that 3E9 anti-MBG Mab effectively lowers BP in hypertensive rats with ESRD, in rats with Ang II-induced hypertension. 3E9 Mab also ex vivo reversed inhibition of the sodium pump in erythrocytes obtained from patients with PE. Humanization of 3E9 Mab is in progress now. (iii) Aging, MBG-induced cell signaling and mechanisms of its modulation. Atrial Natriuretic Peptide (ANP), dephosphoprylates vascular smooth muscle (VSM) NKA via a cGMP-dependent mechanism, and markedly reduces its sensitivity to MBG. In renal medulla of rats, ANP exhibits an opposite effect, i.e., induces NKA phosphorylation and sensitizes the Na pump to the inhibitory effect of MBG. Since VSM and renal medulla express PKG1 and PKG2 isoforms, respectively, these two PKG isoforms are likely to mediate the opposing effects of ANP on NKA phosphorylation and MBG sensitivity. Therefore, a concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, MBG, potentiate each other's natriuretic effects, but ANP may offset the vasoconstrictor effect of MBG. Aging is associated with heightened levels of CTS and with a decline in cGMP-PKG signaling in the kidney and VSM. Such a decline may shift the balance between effects of MBG and ANP on renal and cardiovascular NKA towards lesser inhibition of renal Na pump and greater inhibition of VSM NKA and thus contribute to the pathogenesis of hypertension. In aged Sprague-Dawley rats (S-D), acute NaCl loading produced greater pressor and MBG responses and lesser natriuretic response as compared to that in young rats, We also compared effects of ANP on MBG-induced NKA inhibition in VSM and renal medulla of young and old rats. While in young rats ANP potentiated MBG-induced inhibition of renal NKA, and reversed inhibition of VSM NKA, the effect in aged animals was opposite: ANP did not alter MBG-induced inhibition of renal NKA, and in VSM, ANP potentiated MBG-induced NKA inhibition. Thus, in aged rats, ANP is not capable of potentiation of the physiological effect of MBG, natriuresis, and, likewise, aging is associated with a loss in the ability of ANP to offset MBG-induced vasoconstriction.The phenotype of salt-sensitive hypertension in many aspects resembles that occurring during aging. In our experiments, NaCl loading of salt-sensitive DS and of normotensive S-D, in the presence of comparable MBG response, produces preferential inhibition of the Na pump in the vascular smooth muscle of DS and in the kidney of S-D. Accordingly, NaCl-loaded DS exhibited pressor response and sodium retention, while in S-D adequate natriuresis occurred in the absence of BP elevation. Therefore, in rats, salt-sensitivity of BP is associated with a loss in the responsiveness of renal Na pump to MBG, while vascular Na pump exhibits heightened MBG sensitivity.