University of Southern California, Pharmaceutical Sciences Center, Los Angeles, CA 90089-9121, USA.
Brain Res. 2011 Mar 16;1379:34-43. doi: 10.1016/j.brainres.2011.01.034. Epub 2011 Jan 15.
Previously we demonstrated that 17β-Estradiol (E2) induced rapid Ca(2+) influx via L-type calcium channel activation, which was required for activation of Src/ERK/CREB/Bcl2 signaling cascade and subsequent induction of neuroprotective and neurotrophic responses in rat hippocampal and cortical neurons (Wu et al., 2005; Zhao et al., 2005). The current study determined the presence and specificity of membrane E2 binding sites and the functional consequence of E2 binding to membrane receptors in individual neurons. Using E2-BSA-FITC (fluorescein isothiocyanate) macromolecular complex, membrane E2 binding sites were observed in hippocampal neurons. Punctate FITC signal was observed on plasma membrane of soma and neuronal processes in E2-BSA-FITC binding neurons. No membrane binding was observed with BSA-FITC. Specificity of binding was demonstrated by competition with excess un-conjugated E2. An ERa specific agonist, PPT, and an ERb agonist, DPN, partially competed for E2-BSA-FITC binding. Imaging of intracellular Ca(2+) ([Ca(2+)]i) in live neurons, revealed rapid Ca(2+) responses in E2-BSA-FITC binding neurons within minutes that culminated in a greater [Ca(2+)]i rise and [Ca(2+)]i spikes at >20 min. The same neurons in which E2-BSA-FITC induced a [Ca(2+)]i rise also exhibited activated pERK (extracellular signal-regulated kinase) that was translocated to the nucleus. Immunofluorescent analyses demonstrated that both excitatory and inhibitory neuronal markers labeled subpopulations of E2-BSA-FITC binding neurons. All E2-BSA-FITC binding neurons expressed L-type calcium channels. These results demonstrate, at a single cell level, that E2 membrane receptors mediate the rapid signaling cascades required for E2 neuroprotective and neurotrophic effects in hippocampal neurons. These results are discussed with respect to therapeutic targets of estrogen therapy in brain.
先前我们已经证实，17β-雌二醇（E2）通过 L 型钙通道的激活诱导快速的 Ca（2+）内流，这对于激活Src/ERK/CREB/Bcl2 信号级联以及随后诱导大鼠海马和皮质神经元的神经保护和神经营养反应是必需的（Wu 等人，2005；Zhao 等人，2005）。本研究确定了单个神经元中膜 E2 结合位点的存在和特异性，以及 E2 与膜受体结合的功能后果。使用 E2-BSA-FITC（异硫氰酸荧光素）大分子复合物，观察到海马神经元中的膜 E2 结合位点。在 E2-BSA-FITC 结合神经元的质膜上观察到点状 FITC 信号。BSA-FITC 无膜结合。通过与过量未共轭的 E2 竞争，证明了结合的特异性。ERa 特异性激动剂 PPT 和 ERb 激动剂 DPN 部分竞争 E2-BSA-FITC 结合。在活神经元中对细胞内 Ca（2+）（[Ca（2+）]i）进行成像，发现 E2-BSA-FITC 结合神经元中的 Ca（2+）快速反应在数分钟内发生，最终在 >20 分钟时达到更高的[Ca（2+）]i 升高和[Ca（2+）]i 峰。在 E2-BSA-FITC 诱导 [Ca（2+）]i 升高的相同神经元中，也观察到被激活的 pERK（细胞外信号调节激酶）向核内易位。免疫荧光分析表明，兴奋性和抑制性神经元标记物均标记了 E2-BSA-FITC 结合神经元的亚群。所有 E2-BSA-FITC 结合神经元均表达 L 型钙通道。这些结果在单细胞水平上表明，E2 膜受体介导了 E2 神经保护和神经营养作用所必需的快速信号级联，在海马神经元中。这些结果与大脑中雌激素治疗的治疗靶点有关。
