Miller B A, Foster K, Robishaw J D, Whitfield C F, Bell L, Cheung J Y
Department of Pediatrics, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033.
Blood. 1991 Feb 1;77(3):486-92.
Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.
最近研究表明，源自人类祖细胞的成红细胞对促红细胞生成素（Epo）产生反应时，细胞内游离钙浓度[Ca c]会升高。为了探究鸟苷三磷酸（GTP）结合蛋白在介导[Ca c]升高过程中的作用，将负载有Fura - 2的第10天红系爆式集落形成单位（BFU - E）衍生的单个成红细胞先用百日咳毒素（PT）预处理，再用Epo刺激，然后通过与数字视频成像耦合的荧光显微镜在18分钟内测量[Ca c]。用PT预处理可剂量依赖性地阻断Epo刺激的第10天成红细胞中[Ca c]的升高，但热灭活的PT则无此作用。这些观察结果提供了强有力的证据，表明涉及一种对PT敏感的GTP结合蛋白。为了进一步表征该GTP结合蛋白，将第10天成红细胞膜制剂进行溶解、电泳，并用针对已知对PT敏感的G蛋白亚基的特异性抗体进行免疫印迹：鉴定出了Gia的三种亚型（1、2和3）以及Goa，发现存在Gia1或Gia3以及Gia2，但未发现Goa。为了检测Epo对腺苷酸环化酶活性的影响，先将第10天成红细胞用Epo处理，然后制备分离的膜制剂，并在茶碱存在的情况下测量膜制剂中腺苷酸环化酶产生的环磷酸腺苷（cAMP）。Epo并未抑制反而显著刺激了腺苷酸环化酶活性。然而，[Ca c]升高的机制似乎与腺苷酸环化酶刺激无关，因为用可透过细胞的二丁酰cAMP处理成红细胞未能增加[Ca c]。总之，百日咳毒素可阻断Epo刺激后成红细胞中[Ca c]的升高，这表明该反应是通过一种对百日咳毒素敏感的GTP结合蛋白介导的。在第10天成红细胞上鉴定出的候选对PT敏感的GTP结合蛋白是Gia 1、2或3，但不是Goa。
