Regulation of Neuromodulatory Actions of Angiotensin II in the Brain Neurons by the Ras-Dependent Mitogen-Activated Protein Kinase Pathway

Abstract

Angiotensin II (Ang II) stimulates norepinephrine transporter (NET) and tyrosine hydroxylase (TH) in the neurons, but the signal transduction mechanism of this neuromodulation is not understood. Treatment of neuronal cultures of hypothalamusbrainstem with Ang II resulted in a time-and dose-dependent activation of Ras, Raf-1, and mitogen-activated protein kinase. This activation was mediated by the interaction of Ang II with the AT 1 receptor subtype and was associated with the redistribution of AT 1 receptor with Ras and Raf-1 on the neuronal membrane. Treatment with antisense oligonucleotide (AON) to mitogen-activated protein kinase decreased mitogen-activated protein kinase immunoreactivity by 70% and attenuated Ang II stimulation of c-fos, NET, and TH mRNA levels. This demonstrates that induction of these genes requires mitogenactivated protein kinase activation by Ang II. In contrast, AON to mitogen-activated protein kinase failed to inhibit Ang II stimulation of plasminogen activator inhibitor-1 mRNA levels. These results suggest that AT 1 receptors are coupled to a Ras-Raf-1 mitogen-activated protein kinase signal transduction pathway that is responsible for stimulation of NET and TH, two neuromodulatory actions of Ang II in the brain.

Angiotensin II AT1AReceptor mRNA Expression Is Induced by Estrogen–Progesterone in Dopaminergic Neurons of the Female Rat Arcuate Nucleus

“…Our present results show that this is indeed the case, because we were able to selectively colocalize AT 1A receptor mRNA and tyrosine hydroxylase immunoreactivity in neurons of the arcuate nucleus. It has been shown recently that Ang II can increase tyrosine hydroxylase activity and tyrosine hydroxylase mRNA levels (Yang et al, 1996;Yu et al, 1996). Our present data demonstrate for the first time that tyrosine hydroxylase-producing neurons can express AT 1A receptors and support the hypothesis of the involvement of Ang II in the regulation of neuronal catecholamine synthesis.…”

Involvement of MAP Kinase in Angiotensin II-Induced Phosphorylation and Intracellular Targeting of Neuronal AT1Receptors

“…Lysates were incubated with 1 g of rabbit anti-AT 1 R or anti-MAP kinase antibody overnight at 4ЊC. Immunoprecipitates were collected on protein A/G PLUS-agarose, washed three times with lysis buffer, and used in additional experiments (Yang et al, 1996a).…”

“…Protein-bound antibody was detected by incubation of the membrane with horseradish peroxidase-labeled secondary antibody and enhanced by chemiluminescence assay reagents. The bands recognized by the primary antibody were visualized by exposure to film (Yang et al, 1996a). -Ang II to AT 1 R was determined as follows.…”

“…MAP kinase activity was measured essentially as described elsewhere (Yang et al, 1996a). Briefly, neuronal cells grown in 100-mm-diameter culture dishes were rinsed three times with ice-cold PBS, pH 7.4, and lysed by incubation with 0.5 ml of lysis buffer (25 mM Tris-HCl, 25 mM NaCl, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.5 mM EGTA, 1 mM PMSF, 10 g/ml aprotinin, and 0.8 g/ml leupeptin) for 10 min at 4ЊC.…”

“…In view of this uniqueness of neuronal AT 1 R, we set out to investigate the signal transduction mechanism involved in Ang II regulation of neuromodulation. These studies have revealed a distinct signaling pathway for this GPCR involving Ras-Raf-1 and MAP kinase (Yang et al, 1996a), suggesting that AT 1 R interaction with the heterotrimetric Gq would result in the dissociation of G ␤␥ from G␣. G ␤␥ , then, directly or through the recruitment of one or more cytoplasmic or membrane-associated factors, causes the activation of Ras, which leads to the activation of MAP kinase (Van Corven et al, 1993;Burgering and Bos, 1995;Inglese et al, 1995;Shaw, 1995).…”