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

Na，K-ATP酶由一组负责维持Na+和K+梯度的同工酶组成 穿过大多数细胞的质膜。Na，K-ATPase同工酶多样性的结果来自于 不同分子形式的催化性α和糖基化β亚基的结合， 酵素在这些同工酶中，由α 4同工酶组成的独特的Na，K-ATP酶被选择性地 在睾丸中表达，仅限于雄性生殖细胞。我们已经表明，a4亚型表现出 酶的性质是独特的，这表明多肽发挥作用，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， 经历了显著的发育变化，在配子减数分裂后急剧增加。最 重要的是，抑制A4会损害精子的活动性。这些发现支持了a4在 精子生理学的研究，并表明这种蛋白质是一种有吸引力的避孕目标。Na，K-ATP酶 强心内酯是一组甾体化合物的唯一已知受体。我们 实验室和其他人已经报道，卡登木哇巴因差异性地抑制转运， Na，K-ATP酶同工酶的催化活性，是呈现最高灵敏度的同工酶a4 哇巴因目前，不同于哇巴因的强心内酯对a4的影响尚不清楚。找到 具有更有限的作用并优先抑制A4的化合物对于特异性地 干扰精子中Na+和K+转运。我们的假设是某些强心内酯 抑制α 4超过其它Na，K-ATP酶同工型，并且强心内酯和干扰α 4的化合物 A.4活性可影响精子功能，因此可用作男性避孕药。具体而言，目标 1）鉴定强心内酯类化合物，并通过高通量筛选（HTS）鉴定 影响Na，K-ATP酶α 4亚型的活性，2）确定强心内酯和其他α 4抑制剂的作用 对来自精子的α 4同种型的催化和转运活性的影响，3）为了确定 强心内酯和抑制精子生理学上的A4活性的化合物，以及4）鉴定 A4同种型抑制剂影响精子功能的机制。这项研究将提供新的 男性避孕的药理学方法，并且由于Cardenolides具有可逆作用， 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.