Cardenolides Inhibition of the Sperm Na, K-ATPase Isoiform as Contraceptive

Na，K-ATPase由一组负责维持Na+和K+梯度的同工酶组成 穿过大多数细胞的质膜。Na，K-ATPase的同工酶多样性来自于 不同分子形式的催化a亚基和糖基化p亚基的结合，它们构成 酵素。在这些同工酶中，有一种由OC4亚型组成的独特的Na，K-ATPase是选择性的 在睾丸中表达，仅限于男性生殖细胞。我们已经证明了A4亚型展示了 独特的酶学性质，这表明多肽在维持 雄性生殖细胞的离子梯度、膜电位和兴奋性。此外，我们还发现A4 经历显著的发育变化，在配子减数分裂后急剧增加。多数 重要的是，抑制A4会损害精子的活力。这些发现支持了A4在体内的重要作用。 精子生理学研究表明，这种蛋白质是一种诱人的避孕靶标。Na，K-ATPase是 已知的唯一的卡丹内酯受体，一组本质上是类固醇的化合物。我们的 实验室和其他人已经报道，卡得内酯哇巴因不同程度地抑制细胞的转运和 Na，K-ATPase同工酶的催化活性，A4是表现出最高敏感性的同工酶 为了哇巴因。目前，与哇巴因不同的卡地那内酯对A4的作用尚不清楚。查找 具有更有限的作用并优先抑制A4的化合物将对特定的 干扰精子内Na+和K+的运输。我们的假设是某些卡登内酯优先 抑制A4，而不是其他Na，K-ATPase亚型，以及干扰 A.4活性会影响精子功能，因此可用作男性避孕药。具体地说，目标是 这项建议的主要内容是：1)通过高通量筛选(HTS)鉴定具有以下特性的化合物 对Na，K-ATPase A4亚型活性的影响，2)确定洋甘露内酯等A4抑制剂的作用 精子中OC4异构体的催化和转运活性，3)测定 抑制精子生理学中A4活性的洋茉莉内酯和化合物，以及4)鉴定 A4亚型抑制物影响精子功能的机制。这项研究将提供一种新的 男性避孕的药理学方法，而且由于Cardenolide具有可逆作用，它们 将有助于暂时控制男性生育能力。

The Na,K-ATPase comprises a group of isozymes responsible for maintaining the Na+ and K+ gradients across the plasma membrane of most cells. Isozyme diversity for the Na,K-ATPase results from the association of different molecular forms of the catalytic a and glycosylated p subunits that constitute the enzyme. Among these isozymes, a distinctive Na,K-ATPase composed of the oc4 isoform is selectively expressed in testis, where it is restricted to male germ cells. We have shown that the a4 isoform exhibits enzymatic properties that are unique, which suggested that the polypeptide plays a role in sustaining the ion gradients, membrane potential and excitability of male germ cells. In addition, we have found that a4 undergoes significant developmental changes, increasing dramatically after meiosis of the gametes. Most importantly, inhibition of a4 impairs sperm motility. These findings support an important role for a4 in sperm physiology and show that the protein is an attractive target for contraception. The Na,K-ATPase is the only known receptor for cardenolides, a group of compounds that are steroidal in nature. Our laboratory and others have reported that the cardenolide ouabain differentially inhibits the transport and catalytic activity of the Na,K-ATPase isozymes, being a4 the isoform that presents the highest sensitivity for ouabain. At present, the effect of cardenolides different from ouabain on a4 is unknown. Finding compounds that have a more restricted effect and preferentially inhibit a4 will be important to specifically interfere with Na+ and K+ transport in sperm. Our hypothesis is that certain cardenolides preferentially inhibit a4 over the other Na,K-ATPase isoforms, and that cardenolides and compounds which interfere with a.4 activity can affect sperm function and thus, can be used as male contraceptives. Specifically, the aims of this proposal are: 1) to identify cardenolides and via high throughput screening (HTS) compounds that affect activity of Na,K-ATPase a4 isoform, 2) to establish the effect of cardenolides and other a4 inhibitors on the catalytic and transport activity of the oc4 isoform from spermatozoa, 3) to determine the effect of cardenolides and compounds that inhibit a4 activity on sperm physiology, and 4) to identify the mechanisms by which inhibitors of the a4 isoform affect sperm function. This study will provide a new pharmacological approach to male contraception, and because cardenolides have a reversible effect, they will be useful for the temporary control of male fertility.