Regulation of amphetamine-stimulated dopamine efflux by protein kinase C β
Regulation of amphetamine-stimulated dopamine efflux by protein kinase C β
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DOI：
10.1074/jbc.m413887200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Gnegy, ME
中科院分区：
文献类型：
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作者：
Johnson, LA;Guptaroy, B;Gnegy, ME
有证据表明，蛋白激酶 C (PKC) 和细胞内钙对于安非他明刺激的大鼠纹状体中多巴胺的向外转运非常重要。在这项研究中，我们检查了选定的 PKC 亚型对安非他明刺激的多巴胺流出的影响，重点关注 Ca2+ 依赖性形式的 PKC。在灌注的大鼠纹状体切片中测量内源性多巴胺的流出；多巴胺通过高效液相色谱法测定。非选择性经典 PKC 抑制剂 Go6976 抑制安非他明刺激的多巴胺流出，而 PKC δ 的特异性抑制剂 Rottlerin 则没有效果。高度特异性的 PKC β 抑制剂 LY379196 可阻断由安非他明或 PKC 激活剂 12-O-十四烷酰佛波醇-13-乙酸酯刺激的多巴胺流出。 PKC 抑制剂均未显着改变 [H-3] 多巴胺的摄取。大鼠纹状体膜与多巴胺转运蛋白 (DAT) 共免疫沉淀 PKC beta(I) 和 PKC beta(II)，但不包括 PKC alpha 或 PKC gamma。相反，PKC β(I) 和 PKC β(II) 的抗血清能够共免疫沉淀 DAT，而 PKC α 或 PKC γ 则不能。与单独的 hDAT-HEK 293 细胞或转染 PKC α 或 PKC γ 的 hDAT-HEK 293 细胞相比，转染 PKC beta(II) 的 hDAT-HEK 293 细胞中安非他明刺激的多巴胺流出显着增强。这些结果表明，经典 PKC beta(II) 与 DAT 有物理相关性，并且对于维持大鼠纹状体中安非他明刺激的多巴胺向外转运非常重要。
Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca2+-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Go6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC delta, had no effect. A highly specific PKC beta inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [H-3] dopamine uptake. PKC beta(I) and PKC beta(II), but not PKC alpha or PKC gamma, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC beta(I) and PKC beta(II) but not PKC alpha or PKC gamma were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC beta(II) as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKC alpha or PKC gamma. These results suggest that classical PKC beta(II) is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.