Gil Carles, Najib Abderrahim, Aguilera José
Departament de Bioquímica i de Biologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallés, Barcelona, Spain.
Neurochem Int. 2003 Jun;42(7):535-42. doi: 10.1016/s0197-0186(02)00187-0.
It has been previously shown that 5-HT uptake inhibition produced by tetanus toxin (TeTx) corresponds to a non-competitive inhibition, and it is preceded by phosphorylation of the tyrosine-kinase receptor trkA, phospholipase C activation and translocation of protein kinase C isoforms [FEBS Lett. 481 (2000) 177; FEBS Lett. 486 (2000) 136]. In the present work, it is shown that agonists of tyrosine-kinase receptors (NGF, EGF, basic FGF) enhance Na(+)-dependent, 5-hydroxytryptamine (serotonin, 5-HT) uptake in the synaptosomal-enriched P(2) fraction from rat-brain, suggesting a divergence in the intracellular signal pathways triggered by TeTx and by agonists of TyrK receptors. Co-applications of TeTx and agonists of TyrK receptors result in a mutual and partial reversion of their effects on 5-HT transport. In spite of their differences on transport, TeTx, TPA and NGF produce an increase in serotonin transporter phosphorylation in Ser separately, which is abolished by the PKC-inhibitor bisindolylmaleimide-1. Co-application of sodium vanadate, a tyrosine-phosphatase inhibitor, partially abolishes the effect produced by TeTx, whereas genistein, a tyrosine-kinase inhibitor, does not exert any variation of TeTx inhibition. Analyses by immunoblotting of the activation of specific PKC isoforms activation, determined as translocation to the membrane compartment, reveals differences in the pattern produced by NGF and TeTx. PKC gamma, delta, and epsilon isoforms are equally activated by both compounds, whereas the beta isoform is activated in a sustained manner only by TeTx, and the alpha isoform is only down-regulated by NGF. The aim of the present work was to explore whether NGF have the same effect on 5-HT transport than TeTx, since both compounds share the ability of activate part of the same transduction pathways. In spite of this, growth factors and TeTx show an opposite effect on 5-HT transport, even though SERT phosphorylation is enhanced in both cases. The differential effect on alpha- and beta-PKC isoenzymes found between NGF and TeTx action could explain this apparent discrepancy.
先前的研究表明，破伤风毒素（TeTx）产生的5-羟色胺（5-HT）摄取抑制作用对应于非竞争性抑制，并且在此之前酪氨酸激酶受体trkA会发生磷酸化、磷脂酶C被激活以及蛋白激酶C亚型发生易位[《欧洲生物化学学会联合会快报》481 (2000) 177；《欧洲生物化学学会联合会快报》486 (2000) 136]。在本研究中，结果表明酪氨酸激酶受体激动剂（神经生长因子、表皮生长因子、碱性成纤维细胞生长因子）可增强大鼠脑富含突触体的P(2)组分中依赖Na⁺的5-羟色胺（血清素，5-HT）摄取，这表明TeTx和酪氨酸激酶受体激动剂触发的细胞内信号通路存在差异。TeTx与酪氨酸激酶受体激动剂共同作用会导致它们对5-HT转运的影响相互部分逆转。尽管它们在转运方面存在差异，但TeTx、佛波酯（TPA）和神经生长因子分别会使血清素转运体的丝氨酸磷酸化增加，而这种增加会被蛋白激酶C抑制剂双吲哚马来酰亚胺-1消除。酪氨酸磷酸酶抑制剂钒酸钠共同作用会部分消除TeTx产生的效应，而酪氨酸激酶抑制剂染料木黄酮则不会对TeTx的抑制作用产生任何影响。通过免疫印迹分析特定蛋白激酶C亚型的激活情况（以向膜区室的易位来确定），发现神经生长因子和TeTx产生的模式存在差异。蛋白激酶Cγ、δ和ε亚型被这两种化合物同等激活，而β亚型仅被TeTx持续激活，α亚型仅被神经生长因子下调。本研究的目的是探究神经生长因子对5-HT转运的影响是否与TeTx相同，因为这两种化合物都具有激活部分相同转导通路的能力。尽管如此，生长因子和TeTx对5-HT转运表现出相反的作用，尽管在这两种情况下血清素转运体的磷酸化都有所增强。在神经生长因子和TeTx作用之间发现的对α和β蛋白激酶C同工酶的不同影响可以解释这种明显的差异。
