Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase

Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine -hydroxylase (DH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. PPP3CC levels of TH, DH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148C165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKC and its redistribution from varicosities to neurites is important in Ang IICinduced synaptic accumulation of TH, DH, and NE. They suggest that a coordinated stimulation of transcription of TH, DH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKC-MARCKS pathway are key in persistent stimulation of Ang II’s neuromodulatory actions. (St. Louis, MO). Polyclonal antibodies to TH and DH were obtained from Chemicon (Temecula, CA). Rabbit polyclonal antibodies to various isoforms of PKC (, , ) and agarose-conjugated protein A/G were from (Santa Cruz, CA). Specificity of these antibodies to respective PKC subtypes is more developed by both immunocytochemical staining and by immunoblotting (Asotra and Macklin, 1994; Kim et al., 1994; Sylvia et al., 1996). AntiCrabbit and antiCmouse Fab fragments conjugated with rhodamine or fluorescein had been from (Indianapolis, IN). All the reagents had been bought from (Pittsburgh, PA), and had been the best quality available. Antisense and Feeling oligonucleotides to MARCKS and PKC, PKC, and PKC had been synthesized in the DNA synthesis service from the Interdisciplinary Middle for Biotechnology Study, College or university of Florida (Gainesville, FL). These sequences 717907-75-0 have already been used effectively to downregulate MARCKS and PKC by additional organizations (Aigner and Caroni, 1993; Balboa et 717907-75-0 al., 1994). The sequences are detailed the following: MARCKS: Feeling: 5-AAGAAGCCAGCATGGGTGCACAGTT-3 Antisense: 5-AACTGTGCACCCATGCTGGCTTCTT-3 PKC: Feeling: 5-GAACCATGGCTGACGTTTACC-3 Antisense: 5-GGTAAACGTCAGCCATGGTTC-3 PKC: Feeling: 5-AAGATGGCTGACCCGGCTCGC-3 Antisense: 5-CGCAGCCGGGTGACCCGGCCGC-3 PKC: Feeling: 5-CAAGATGGCTGACCCGGCCGC-3 Antisense: 5-GCGGCCGGGTCAGCCATCTTG-3 A artificial peptide related to proteins 148C165 of MARCKS (KRFSFKKSFKLSGFSFKK; pep148C165) and its own mutant counterpart where serines at positions 151, 155, 159, and 717907-75-0 162, had been replaced by alanine (KRFAFKKAFKLAGFAFKK; mut148C165) had been synthesized by Genemed Biotechnologies (SAN FRANCISCO BAY AREA, CA). Pep148C165 competes for PKC-mediated phosphorylation of MARCKS since three from the four serines (151, 155, and 162) with this molecule are known PKC phosphorylation sites (Amess et al., 1992). On the other hand, mut148C165 wouldn’t normally be a rival, and acts as control for pep148C165 as a result. Planning of Neuronal Ethnicities HypothalamusCbrainstem regions of 1-d-old WKY rat brains had been dissected and mind cells had been dissociated by trypsin as referred to previously (Raizada et al., 1984, 1993). Dissociated mind cells had been plated in poly- l-lysineCprecoated cells culture meals (2 107 cells/100-mm-diam dish or 3 106 cells/35-mm-diam dish) 717907-75-0 in DME including 10% PDHS and neuronal tradition founded as previously referred to (Raizada et al., 1984, 1993). The ethnicities had been allowed to develop for 15 d before their make use of in tests. These ethnicities contain 85C90% neuronal cells and 10C15% astroglial cells (Raizada et al., 1984, 1993). Dedication of TH, DH Immunoreactivities, and Particular [3H]NE Uptake in Synaptosomal Arrangements of Neuronal Cells Neuronal cells, founded in 100-mm-diam cells culture dishes, had been subjected to various pretreatments followed by incubation with 100 nM Ang II for indicated time periods. Cells from 10 culture dishes were collected, cell pellets homogenized in 0.32 M sucrose, and then homogenates were used for synaptosomal preparation essentially as described elsewhere for neuronal cultures (Kishi et al., 1991). Purity of the synaptosomal fraction was established with the use of synaptophysin antibody as marker (Kishi et al., 1991). Synaptosomal preparations containing 100 g protein were subjected to 4C15% SDS-PAGE, separated proteins were transferred to nitrocellulose membrane, and then blotted with the use of 1 g/ml anti-TH or anti-DH antibodies essentially as described previously (Lu et al., 1997). Antibodies bound to TH or DH were identified by HRP-labeled antiCrabbit antibody and visualized by chemiluminescence as described previously (Yang et al., 1996; Lu et al., 1997). Bands corresponding to TH and DH immunoreactivities were quantitated by SW5000 Gel Analyzer after ascertaining that densities of each immunoreactive band was within the linear range as described.