Black J A, Dib-Hajj S, Cohen S, Hinson A W, Waxman S G
Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, USA.
Glia. 1998 Jul;23(3):200-8.
Astrocytes in vitro express several distinct voltage-sensitive sodium currents, including tetrodotoxin (TTX)-resistant in non-stellate astrocytes and TTX-sensitive currents in stellate astrocytes. However, the molecular identity of the underlying channels, and the mechanisms that regulate their expression, have yet to be identified. Since spinal cord astrocytes in vitro express sodium currents that are nearly ten-fold greater that those of astrocytes derived from other regions, we used reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunocytochemistry to search for a sodium channel mRNA and protein corresponding to a TTX-resistant channel in these cells. RT-PCR did not detect transcripts for SNS, which is known to encode a TTX-resistant current in dorsal root ganglion neurons. However, RT-PCR demonstrated the presence of rH1 mRNA in cultured spinal cord astrocytes derived from postnatal day 0 (P0) Sprague Dawley rats at 7 days in vitro and in also intact spinal cords of P0 and P7 rats. Hybridization signal for rH1 mRNA was detected by in situ hybridization cytochemistry in most non-stellate and, at varying levels, in stellate astrocytes in these cultures. Immunocytochemical studies, utilizing a polyclonal antibody (R-12) generated against a conserved polypeptide sequence of sodium channels, demonstrated sodium channel immunoreactivity in non-stellate and stellate astrocytes in these cultures. Spinal cord cultures reacted with a rH1-specific polyclonal antibody also showed rH1 immunostaining in non-stellate and stellate astrocytes, although the intensity of the rH1 immunoreactivity in both astrocyte morphologies was attenuated compared to that observed with the R-12 generic sodium channel antibody. The presence of rH1 mRNA and protein in non-stellate astrocytes in vitro provides a possible correlate for the TTX-resistant current that has been recorded in these cells. Since TTX-resistant current is not present in stellate astrocytes, the presence of rH1 mRNA and protein in these cells suggests, in addition, that post-translational mechanisms participate in the control of sodium channel expression in these cells.
体外培养的星形胶质细胞表达几种不同的电压敏感性钠电流，包括非星状星形胶质细胞中的河豚毒素（TTX）抗性电流和星状星形胶质细胞中的TTX敏感性电流。然而，潜在通道的分子特性以及调节其表达的机制尚未明确。由于体外培养的脊髓星形胶质细胞表达的钠电流比源自其他区域的星形胶质细胞的钠电流大近十倍，我们使用逆转录聚合酶链反应（RT-PCR）、原位杂交和免疫细胞化学方法，来寻找与这些细胞中TTX抗性通道相对应的钠通道mRNA和蛋白质。RT-PCR未检测到SNS的转录本，已知SNS在背根神经节神经元中编码一种TTX抗性电流。然而，RT-PCR证明在体外培养7天的出生后第0天（P0）的Sprague Dawley大鼠的脊髓星形胶质细胞以及P0和P7大鼠的完整脊髓中存在rH1 mRNA。通过原位杂交细胞化学在这些培养物中的大多数非星状星形胶质细胞以及不同程度地在星状星形胶质细胞中检测到rH1 mRNA的杂交信号。利用针对钠通道保守多肽序列产生的多克隆抗体（R-12）进行的免疫细胞化学研究表明，在这些培养物的非星状和星状星形胶质细胞中存在钠通道免疫反应性。与rH1特异性多克隆抗体反应的脊髓培养物在非星状和星状星形胶质细胞中也显示出rH1免疫染色，尽管与用R-12通用钠通道抗体观察到的相比，两种星形胶质细胞形态中rH1免疫反应性的强度都有所减弱。体外非星状星形胶质细胞中rH1 mRNA和蛋白质的存在为这些细胞中记录到的TTX抗性电流提供了一种可能的关联。由于星状星形胶质细胞中不存在TTX抗性电流，这些细胞中rH1 mRNA和蛋白质的存在还表明，翻译后机制参与了这些细胞中钠通道表达的控制。
