Schmitt B M, Berger U V, Douglas R M, Bevensee M O, Hediger M A, Haddad G G, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
J Neurosci. 2000 Sep 15;20(18):6839-48. doi: 10.1523/JNEUROSCI.20-18-06839.2000.
We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.
我们使用源自电生性大鼠肾钠/碳酸氢根共转运体(rkNBC)的多核苷酸探针和多克隆抗体,研究了钠/碳酸氢根共转运体在大鼠脑中的表达和分布。在全脑中,通过Northern杂交我们观察到一条单一的mRNA(约7.5 kb),并用针对rkNBC C末端的多克隆抗血清进行免疫印迹检测到一条主要的约130 kDa的蛋白条带。钠/碳酸氢根共转运体(NBC)的mRNA和蛋白存在于皮质、脑干-间脑和小脑中。原位杂交显示整个中枢神经系统(CNS)均有NBC mRNA表达,在嗅球、海马齿状回和小脑中表达水平尤其高。NBC mRNA存在于神经胶质细胞(如小脑的伯格曼胶质细胞和海马星形胶质细胞)和神经元(如齿状回的颗粒细胞以及皮质或纹状体的神经元)中。对编码NBC的mRNA与谷氨酸转运体1(神经胶质细胞标志物)进行双重杂交证实,神经胶质细胞和神经元均表达NBC。间接免疫荧光显微镜检查显示整个中枢神经系统均有NBC蛋白,特别是在海马和小脑中。尽管NBC mRNA局限于细胞体,但NBC蛋白分布弥散,这与它在细胞突起甚至可能在细胞体中的定位相符。用神经胶质纤维酸性蛋白(星形胶质细胞标志物)、微管相关蛋白2(神经元标志物)或2',3'-环磷酸核苷酸3'-磷酸二酯酶(少突胶质细胞标志物)进行双重标记显示,神经胶质细胞和神经元的特定亚群均表达NBC蛋白。此外,培养的海马星形胶质细胞和皮质神经元中也存在NBC蛋白。脉络丛、室管膜和脑膜的上皮细胞中也存在NBC mRNA和蛋白。因此,我们的结果与钠/碳酸氢根共转运在中枢神经系统生理学中的多种新作用相一致。