Lyng F M, Jones G R, Rommerts F F
Daresbury Laboratory, Daresbury, Warrington, Cheshire WA4 4AD, United Kingdom.
Biol Reprod. 2000 Sep;63(3):736-47. doi: 10.1095/biolreprod63.3.736.
Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2-3 min duration) increase in intracellular calcium levels was observed within 20-30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1-1000 pM induced transient calcium increases with ED(50) values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED(50) values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17beta were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.
在三种不同的细胞类型中研究了雄激素诱导的钙通量和间隙连接细胞间通讯（GJIC）。在添加雄激素后20 - 30秒内观察到细胞内钙水平短暂（持续2 - 3分钟）升高，随后在类固醇浓度高于1 nM时进入平台期。未成熟大鼠支持细胞（含有正常核受体）、人前列腺肿瘤细胞系LNCaP（含有突变核受体）和人前列腺细胞系PC3（不含有核受体）中钙反应的动力学相似。人A431肿瘤细胞系对雄激素无反应。睾酮和合成雄激素R1881浓度在1 - 1000 pM之间可诱导钙短暂升高，ED(50)值接近1 pM和1 nM，而双氢睾酮（DHT）在这些浓度下无活性。在浓度高于1 nM时，睾酮、R1881和DHT在刺激持续超过10分钟的钙升高方面等效，ED(50)值在5至20 nM之间。与白蛋白共价结合的睾酮也有活性，而11种相关雄烷化合物以及孕酮和雌二醇 - 17β在1000 nM时无活性。羟基氟他胺（1000 nM）和非那雄胺（1000 nM）可消除所有细胞类型中三种雄激素（10 nM）诱导的钙反应，但醋酸环丙孕酮（1000 nM）不能。在无细胞外钙的情况下钙反应也被消除，且维拉帕米的存在可强烈抑制该反应。将反应性细胞暴露于短暂（150秒）的雄激素脉冲产生的钙反应与持续暴露后的反应相似。支持细胞仅暴露于100 nM睾酮30秒后，钙反应持续至少50分钟。尽管可证明雄激素与细胞核结合，但在类似条件下没有证据表明其与质膜紧密结合。当蛋白质合成被抑制时，在添加10 nM睾酮后15分钟内观察到大鼠支持细胞之间GJIC增强，但LNCaP细胞或PC3细胞之间未增强。由于PC3细胞中不存在核雄激素，且反应系统的许多功能特性在所有三种细胞类型中均与核受体不同，我们推测存在一种具有双相反应特征（高亲和力和低亲和力）的替代性细胞表面受体系统。钙信号可能与支持细胞之间间隙连接效率的调节相关。低亲和力受体可能在局部水平升高时（如在睾丸中，当核受体（过度）饱和时）传递互补的雄激素信号。
