Jaiswal Bijay S, Conti Marco
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10676-81. doi: 10.1073/pnas.1831008100. Epub 2003 Sep 4.
In mammals, Ca2+ and HCO3- ions play a critical role in the regulation of sperm function, most likely by regulation of cAMP levels. Mammalian germ cells contain a soluble adenylyl cyclase (sAC) with properties distinct from the well characterized membrane-bound enzymes Here we investigated whether the cyclase expressed in mature spermatozoa has the properties of sAC and whether it is regulated by Ca2+. In addition to an HCO3--dependent activation, the cyclase endogenous to human spermatozoa is stimulated 2- to 3-fold by Ca2+ in a concentration-dependent manner (EC50 approximately 400 nM). In a similar fashion, Ca2+ activates the recombinant rat and human full-length sAC with similar EC50 values. The Ca2+ stimulation was also observed when sAC was activated with HCO3-, was independent of calmodulin, and was associated with an increase in Vmax without changes in Km for ATP-Mg2+. An increase in intracellular Ca2+ by ionophore or by a muscarinic cholinergic receptor agonist increases cAMP in cells transfected with FL-hsAC, but not in mock-transfected cells. Similarly, both Ca2+ and HCO3- stimulate cAMP accumulation in human spermatozoa. These findings provide evidence that human spermatozoa express a cyclase with the properties of sAC and that Ca2+ can substitute for HCO3- in the stimulation of this enzyme, underscoring an important role for sAC in the control of sperm functions.
在哺乳动物中,Ca2+和HCO3-离子在精子功能调节中起关键作用,很可能是通过调节环磷酸腺苷(cAMP)水平来实现的。哺乳动物生殖细胞含有一种可溶性腺苷酸环化酶(sAC),其特性与特征明确的膜结合酶不同。在此,我们研究了成熟精子中表达的环化酶是否具有sAC的特性,以及它是否受Ca2+调节。除了依赖HCO3-的激活外,人类精子内源性环化酶还能被Ca2+以浓度依赖的方式刺激2至3倍(半数有效浓度约为400 nM)。以类似方式,Ca2+以相似的半数有效浓度激活重组大鼠和人类全长sAC。当用HCO3-激活sAC时也观察到Ca2+刺激,该刺激不依赖钙调蛋白,且与Vmax增加相关,而ATP-Mg2+的米氏常数(Km)不变。通过离子载体或毒蕈碱胆碱能受体激动剂使细胞内Ca2+增加,可使转染全长人可溶性腺苷酸环化酶(FL-hsAC)的细胞中cAMP增加,但在mock转染细胞中则不然。同样,Ca2+和HCO3-均刺激人类精子中的cAMP积累。这些发现提供了证据,表明人类精子表达具有sAC特性的环化酶,且Ca2+在刺激该酶时可替代HCO3-,强调了sAC在控制精子功能中的重要作用。