Cook S P, Babcock D F
Department of Biochemistry, University of Washington, Seattle 98195.
J Biol Chem. 1993 Oct 25;268(30):22402-7.
The egg peptide speract stimulates sperm guanylyl cyclase and presumably enhances fertilization, but the roles of cGMP in sperm responses are yet undetermined. Here we show that speract-induced accumulation of cGMP or cAMP is selectively enhanced by the phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (IBMX) or papaverine, respectively. These inhibitors provided the unusual opportunity to examine the consequences of manipulating cGMP- and cAMP-dependent responses. The following observations suggest that cGMP mediates activation of K channels, the earliest known ionic event in speract signal transduction: 1) both cGMP content and K+ permeability are maximal within 15 s of speract stimulation and both decline after intracellular pH (pHi) increases in response to hyperpolarization; 2) IBMX prolongs elevation of cGMP and sustains K+ permeability after pHi increases; 3) both cGMP accumulation and K+ permeability also are enhanced when the pHi increase is prevented by an elevated concentration of external K+ (Ko); 4) elevating pHi with NH4Cl bypasses the blockade imposed by high Ko and decreases K+ permeability. Because IBMX antagonizes this action of NH4Cl, these results further suggest that elevation of pHi initiates an inactivation of guanylyl cyclase that leads to K channel closure. However, K+ permeability is restored upon subsequent elevation of intracellular [Ca2+] (Cai), indicating either that sperm K channels possess an alternate regulatory mode, or that a distinct Ca(2+)-activated K permeability also participates in speract signal transduction. Regardless of the mechanism that mediates Cai action, sperm K channels are identified as downstream targets of cGMP and are implicated in a feedback loop that both terminates guanylyl cyclase activity and leads to their own inactivation.
卵肽精子活化肽刺激精子鸟苷酸环化酶,可能会增强受精作用,但cGMP在精子反应中的作用尚未确定。在此我们表明,磷酸二酯酶抑制剂3 -异丁基-1 -甲基黄嘌呤(IBMX)或罂粟碱分别选择性地增强了精子活化肽诱导的cGMP或cAMP积累。这些抑制剂提供了一个特殊的机会来研究操纵cGMP依赖性和cAMP依赖性反应的后果。以下观察结果表明,cGMP介导钾通道的激活,这是精子活化肽信号转导中最早已知的离子事件:1)在精子活化肽刺激后15秒内,cGMP含量和钾离子通透性均达到最大值,并且在细胞内pH值(pHi)因超极化而升高后两者均下降;2)IBMX延长了cGMP的升高,并在pHi升高后维持钾离子通透性;3)当细胞外钾离子(Ko)浓度升高阻止pHi升高时,cGMP积累和钾离子通透性也会增强;4)用氯化铵升高pHi可绕过高Ko施加的阻断并降低钾离子通透性。由于IBMX拮抗氯化铵的这种作用,这些结果进一步表明pHi升高引发鸟苷酸环化酶失活,导致钾通道关闭。然而,随后细胞内钙离子(Cai)浓度升高时钾离子通透性恢复,这表明要么精子钾通道具有另一种调节模式,要么一种独特的钙激活钾离子通透性也参与精子活化肽信号转导。无论介导Cai作用的机制如何,精子钾通道被确定为cGMP的下游靶点,并参与一个反馈回路,该回路既能终止鸟苷酸环化酶活性,又能导致其自身失活。