Lu D, Yang H, Lenox R H, Raizada M K
Department of Physiology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
J Cell Biol. 1998 Jul 13;142(1):217-27. doi: 10.1083/jcb.142.1.217.
Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. These neuromodulatory actions of Ang II involve Ras-Raf-MAP kinase signal transduction pathways (Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609-1617). In this study, we present evidence to demonstrate participation of another signaling pathway in these neuronal actions of Ang II. It involves activation of protein kinase C (PKC)beta subtype and phosphorylation and redistribution of myristoylated alanine-rich C kinase substrate (MARCKS) in neurites. Ang II caused a dramatic redistribution of MARCKS from neuronal varicosities to neurites. This was accompanied by a time-dependent stimulation of its phosphorylation, that was mediated by the angiotensin type 1 receptor subtype (AT1). Incubation of neurons with PKCbeta subtype specific antisense oligonucleotide (AON) significantly attenuated both redistribution and phosphorylation of MARCKS. Furthermore, depletion of MARCKS by MARCKS-AON treatment of neurons resulted in a significant decrease in Ang II-stimulated accumulation of TH and DbetaH immunoreactivities and [3H]NE uptake activity in synaptosomes. In contrast, mRNA levels of TH, DbetaH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148-165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DbetaH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKCbeta and its redistribution from varicosities to neurites is important in Ang II-induced synaptic accumulation of TH, DbetaH, and NE. They suggest that a coordinated stimulation of transcription of TH, DbetaH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKCbeta-MARCKS pathway are key in persistent stimulation of Ang II's neuromodulatory actions.
血管紧张素II（Ang II）对酪氨酸羟化酶（TH）、多巴胺β羟化酶（DbetaH）和去甲肾上腺素转运体（NET）具有慢性刺激作用，部分是通过影响它们基因的转录来实现的。Ang II的这些神经调节作用涉及Ras-Raf-MAP激酶信号转导途径（Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609-1617）。在本研究中，我们提供证据证明另一种信号通路参与了Ang II的这些神经元作用。它涉及蛋白激酶C（PKC）β亚型的激活以及神经突中富含肉豆蔻酰化丙氨酸的C激酶底物（MARCKS）的磷酸化和重新分布。Ang II导致MARCKS从神经元膨体显著重新分布到神经突。这伴随着其磷酸化的时间依赖性刺激，这是由1型血管紧张素受体亚型（AT1）介导的。用PKCβ亚型特异性反义寡核苷酸（AON）孵育神经元可显著减弱MARCKS的重新分布和磷酸化。此外，用MARCKS-AON处理神经元使MARCKS耗竭，导致Ang II刺激的突触体中TH和DbetaH免疫反应性积累以及[3H]NE摄取活性显著降低。相比之下，TH、DbetaH和NET的mRNA水平不受MARKS-AON处理的影响。包含PKC磷酸化位点的MARCKS pep148-165抑制了Ang II对MARCKS磷酸化的刺激，并减少了神经元突触体中TH、DbetaH和[3H]NE的摄取量。这些观察结果表明，PKCβ介导的MARCKS磷酸化及其从膨体到神经突的重新分布在Ang II诱导的TH、DbetaH和NE的突触积累中很重要。它们表明，由Ras-Raf-MAP激酶介导的TH、DbetaH和NET转录的协同刺激，随后由PKCβ-MARCKS途径介导它们的转运，是Ang II持续神经调节作用的关键。
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