Supplementary Material for: Pannexin Protein Expression in the Rat Middle Cerebral Artery

<i>Background:</i> Connexin proteins are well known to participate in cell-to-cell communication within the cerebral vasculature. Pannexins are a recently discovered family of proteins that could potentially be involved in cell-to-cell communication. Herein, we sought to determine whether pannexins are expressed in rat middle cerebral artery (MCA). <i>Methods:</i> A combination of RT-PCR, immunoblotting and immunohistochemistry techniques was used to characterize the expression pattern of pannexins in rat MCA. A fluorescent dye uptake approach in cultured smooth muscle cells was used to determine whether these cells have functional hemichannels. <i>Results:</i> We report for the first time that pannexins are expressed in the cerebral vasculature. We reveal that pannexin 1 is expressed in smooth muscle but not in endothelium and pannexin 2 is expressed in both endothelium and smooth muscle. Fluorescent dye entered cultured smooth muscle cells in the absence of extracellular calcium or when the cells were depolarized, which was prevented by the putative hemichannel blocker carbenoxolone. <i>Conclusions:</i> The identification of pannexins in rat MCA indicates that pannexin expression is not restricted to neuronal cells. Dye uptake in cultured smooth muscle cells exhibited properties similar to those of connexin and pannexin hemichannels, which may represent another form of cell-to-cell communication within the vasculature.

<背景：> 连接蛋白（Connexin）是一类公认参与脑血管系统细胞间通讯的蛋白。泛连接蛋白（Pannexin）是近年发现的蛋白家族，其可能参与细胞间通讯。本研究旨在明确泛连接蛋白是否在大鼠大脑中动脉（MCA）中表达。
<方法：> 本研究联合采用逆转录聚合酶链反应（RT-PCR）、免疫印迹（immunoblotting）及免疫组织化学（immunohistochemistry）技术，对泛连接蛋白在大鼠大脑中动脉中的表达模式进行表征。同时通过培养平滑肌细胞的荧光染料摄取法，检测这些细胞是否具备功能性半通道（hemichannel）。
<结果：> 本研究首次证实泛连接蛋白在脑血管系统中表达。研究发现，泛连接蛋白1（Pannexin 1）仅表达于平滑肌细胞，而不表达于内皮细胞；泛连接蛋白2（Pannexin 2）则同时表达于内皮细胞与平滑肌细胞。在无细胞外钙或细胞发生去极化的情况下，荧光染料可进入培养的平滑肌细胞，而这一过程可被半通道的潜在阻滞剂甘珀酸（carbenoxolone）所阻断。
<结论：> 大鼠大脑中动脉中泛连接蛋白的鉴定结果表明，泛连接蛋白的表达并非仅限于神经元细胞。培养平滑肌细胞的染料摄取实验显示，其通道特性与连接蛋白及泛连接蛋白半通道相似，这可能代表了血管系统内另一种形式的细胞间通讯方式。