Komoly S, Liu Y, Webster H D, Chan K F
Laboratory of Experimental Neuropathology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J Neurosci Res. 1991 Jul;29(3):379-89. doi: 10.1002/jnr.490290313.
Light (LM) and electron (EM) microscopic immunocytochemical methods were used to study the distribution of protein kinase C (PKC) isozymes in adult rat optic nerves. In cryostat and vibratome sections examined by LM, type II (beta) isozyme was localized almost exclusively in the axons. In the EM, immunoreaction products were found to associate with microtubules and neurofilaments. The inner surface of axonal membranes were occasionally stained. Analysis of PKC isozyme composition of the optic nerves by using immunoblot techniques revealed that type II (beta) isozyme accounted for approximately 80% of the total immunoreactivity. By contrast, type III (alpha) isozyme, which accounted for the remaining 20% of PKC, was found mainly in the astrocytes. Astrocytic processes next to blood vessels and between myelinated axons were stained. In the EM, immunoreaction products were found in the cytoplasm and along astroglial filaments. Segments of plasma membranes also were stained; but nuclei were unstained. Adult glial cells were not stained by an antibody to type II (beta) isozyme except for the occurrence of a few punctate cytoplasmic densities in occasional astrocytes. Very faint or no immunostaining was observed in sections treated with a monoclonal antibody to type I (gamma) isozyme. Immunoblot analyses also did not reveal this subspecies. The absence of type I (gamma) isozyme in optic nerves is not due to a down-regulation of the enzyme during development. In developing (5 and 11 day) rats, immunoreactivity of protein kinase C was very faint or absent. After 15 days, reaction products of both type III (alpha) and type II (beta) isozymes were found throughout the nerve. These findings suggest that type II (beta) isozyme may be involved in axonal transport whereas type III (alpha) isozyme may play a role in some astrocyte functions in mature optic nerves.
采用光学显微镜（LM）和电子显微镜（EM）免疫细胞化学方法研究成年大鼠视神经中蛋白激酶C（PKC）同工酶的分布。在光学显微镜检查的低温恒温器切片和振动切片机切片中，II型（β）同工酶几乎仅定位于轴突中。在电子显微镜下，发现免疫反应产物与微管和神经丝相关。轴突膜的内表面偶尔会被染色。利用免疫印迹技术分析视神经的PKC同工酶组成，结果显示II型（β）同工酶约占总免疫反应性的80%。相比之下，占PKC其余20%的III型（α）同工酶主要存在于星形胶质细胞中。血管旁和有髓轴突之间的星形胶质细胞突起被染色。在电子显微镜下，免疫反应产物存在于细胞质中并沿着星形胶质细胞丝分布。质膜片段也被染色，但细胞核未被染色。除了偶尔在星形胶质细胞中出现一些点状细胞质致密物外，成年神经胶质细胞未被II型（β）同工酶抗体染色。在用I型（γ）同工酶单克隆抗体处理的切片中观察到非常微弱或没有免疫染色。免疫印迹分析也未发现该亚类。视神经中I型（γ）同工酶的缺失并非由于该酶在发育过程中的下调。在发育中的（5日龄和11日龄）大鼠中，蛋白激酶C的免疫反应性非常微弱或不存在。15天后，在整个神经中发现III型（α）和II型（β）同工酶的反应产物。这些发现表明，II型（β）同工酶可能参与轴突运输，而III型（α）同工酶可能在成熟视神经的某些星形胶质细胞功能中发挥作用。
