Apparsundaram S, Schroeter S, Giovanetti E, Blakely R D
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
J Pharmacol Exp Ther. 1998 Nov;287(2):744-51.
Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, beta-PMA or beta-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km. Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, beta-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetine Kd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.
在神经元质膜中发现的去甲肾上腺素（NE）转运体（NETs）介导从细胞外空间清除NE，限制去甲肾上腺素能突触处肾上腺素能受体的激活。我们先前对去甲肾上腺素能神经母细胞瘤SK-N-SH的研究揭示了毒蕈碱受体触发的NET表面密度和转运能力的调节，部分由蛋白激酶C激活介导。然而，在这个天然细胞模型中NET蛋白丰度较低，妨碍了对NET蛋白转运改变的直接确认。在我们的研究中，我们监测了激酶激活剂和抑制剂作用后，瞬时和稳定转染细胞系中人类NET蛋白的活性和表面分布。使用稳定转染hNET的HEK-293和LLC-PK1细胞，以及瞬时转染的COS-7细胞，我们证明蛋白激酶C激活剂佛波酯、β-PMA或β-PDBu选择性降低l-NE转运能力（Vmax），而NE Km变化不大。佛波酯的作用迅速、具有立体特异性，并被蛋白激酶C抑制剂星形孢菌素和双吲哚马来酰亚胺I阻断。与SK-N-SH细胞一样，β-PMA导致完整细胞[3H]尼索西汀结合位点减少，而尼索西汀Kd或总膜NET密度无变化。细胞表面生物素化和共聚焦免疫荧光技术证实，蛋白激酶C依赖性的NE转运能力和全细胞拮抗剂结合密度降低伴随着细胞表面人类NET蛋白表达的降低。这些发现共同表明，激酶调节的蛋白转运是抗抑郁药敏感的NE清除急性调节的潜在途径。
