Angiotensin II Increases Vesicular Trafficking in Live Noradrenergic Brain Neurons

Abstract

P1 Angiotensin II (Ang II) interacts with the AT 1 receptor subtype and stimulates turnover of norepinephrine (NE) by regulating its synthesis, release, and uptake. The cellular mechanism by which Ang II regulates this neurotransmission is not well understood. The aim of this study was to test the hypothesis that Ang II increases vesicular trafficking to increase synaptic release of NE. cDNA encoding full length rat dopamine beta-hydroxylase (DβH) fused to Green Fluorescence Protein (GFP) was cloned in pCl-Neo vector under the control of CMV promoter and used to tag synaptic vesicles. Transfection of cells with this plasmid resulted in the expression of green fluorescence and GFP-DβH fusion protein which was functional as judged by an increase in DβH activity. Transfected neurons expressed bright green fluorescence representing GFP-DβH that was predominantly localized in the cell soma. Few neurites also displayed the fluorescence. Real time confocal microscopic examination of live neurons indicated that Ang II caused a time-dependent increase in both the intensity and the number of neurites depicting green fluorescence. An increase in GFP-DβH in the neurites became apparent within one minute and persisted for 15-20 minutes. A 2.5 fold increase in the GFP-DβH positive neurites was observed by 100 nM Aug II in 15 min [control, 2±0.5 (n=20) versus Ang II treatment, 5±0.4 (n=25)]. Ang II-induced GFP-DβH redistribution was punctate, co-localized with synaptophysin and was blocked by losartan, and not by PD123319. Release of Ang II induced NE paralleled its effect on GFP-DβH distribution. PMA treatment caused translocation of GFP-DβH in a fashion comparable with Ang II. In addition, inactivation of protein kinase C by a prolonged incubation of neurons with PMA, completely blocked the translocation of GFP-DβH from the cell body to neurites. These observations demonstrate that Ang II stimulates trafficking of NE vesicles by a PKC-dependent mechanism. Thus, an increased trafficking of NE containing vesicles from the neuronal cell body to the neurites may be key to Ang II stimulated release of NE from a noradrenergic neuron.