Angiotensin II (AII) acts at neuronal receptors in the hypothalamus and brainstem to stimulate numerous physiological effects including increased blood pressure, fluid intake and vasopressin secretion, and also possibly neurotrophic actions (either direct or by modulation of the effects of growth factors). The overall aim of this proposal is to investigate the characteristics, intracellular coupling, cellular functions and regulation of AII receptor subtypes in neurons cultured from rat hypothalamus and brainstem. These brain areas contain AT1- receptors (AT1-R) and AT2-receptors (AT2-R), and current knowledge of the cellular mechanisms mediated by these receptors in neurons, and also their regulation, is rudimentary. Our published studies, preliminary data and also the background literature are consistent with the following hypotheses: Neurons contain AT1-R which are coupled to a stimulation of inositol phospholipid (IP) hydrolysis, with subsequent Ca2+ mobilization and protein kinase C (PKC) activation. Further, these AT1-R mediate the effects of AII on a variety of cellular functions, including: (a) PKC-and Ca2+-dependent stimulation of norepinephrine (NE) synthesis, release and metabolism; (b) PKC-dependent increases in d- glucose transport into neurons, mediated via a cascade of events that includes stimulation of c-fos mRNA, c-fos, and glucose transporter 3 (Glut-3) mRNA. Neurons also contain AT2-R, which are coupled to increased Ca2+ entry, with subsequent activation of a phosphodiesterase (PDE) and a decrease in cGMP, but the cellular functions of these AT2-R are unknown. Lastly, modulators of neuronal AII receptors and responses (e.g., NE, aldosterone [ALDO] show selective regulatory effects for each neuronal AII receptor subtype. In the studies proposed here we plan to investigate each of the above hypotheses, and also determine the binding characteristics (levels/affinities) of each neuronal AII receptor subtype. The present studies are thus designed to investigate the cell physiology of AT1-R and AT2-R in neurons. The significance of the studies is that we will identify mechanisms (i.e., AII receptor subtype/intracellular event/cellular function/receptor regulation) which are involved in the physiological actions mediated by neuronal AII receptors. This fundamental information is for a full understanding of the CNS-mediated actions of AII on the cardiovascular system and fluid intake (both of which involve neuronal NE) and also neurotrophic effects of this peptide (which involve c-fos and d-glucose transport). In the long term this information will be valuable for determining the mechanism which underlie abnormalities in AII effects in the CNS, e.g., the increased expression and responsiveness of neuronal AII receptors which occur in certain forms of hypertension.
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