Abstract
Angiotensin II (AngII) acts on central neurons to increase neuronal firing and induce sympathoexcitation, which contribute to the pathogenesis of cardiovascular diseases including hypertension and heart failure. Numerous studies have examined the precise AngII-induced intraneuronal signaling mechanism in an attempt to identify new therapeutic targets for these diseases. Considering the technical challenges in studying specific intraneuronal signaling pathways in vivo, especially in the cardiovascular control brain regions, most studies have relied on neuronal cell culture models. However, there are numerous limitations in using cell culture models to study AngII intraneuronal signaling, including the lack of evidence indicating the stability of AngII in culture media. Herein, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Using liquid chromatography-tandem mass spectrometry, we measured levels of AngII and its metabolites, Ang III, Ang IV, and Ang-1-7, in neuronal cell culture media after administration of exogenous AngII (100 nmol/L) to a neuronal cell culture model (CATH.a neurons). AngII levels rapidly declined in the media, returning to near baseline levels within 3 h of administration. Additionally, levels of Ang III and Ang-1-7 acutely increased, while levels of Ang IV remained unchanged. Replenishing the media with exogenous AngII every 3 h for 24 h resulted in a consistent and significant increase in AngII levels for the duration of the treatment period. These data indicate that AngII is rapidly metabolized in neuronal cell culture media, and replenishing the media at least every 3 h is needed to sustain chronically elevated levels.
Highlights
Dysregulation of angiotensin II (AngII)-dependent neuronal signaling in the central nervous system is involved in the pathogenesis of cardiovascular diseases, such as hypertension and chronic heart failure (Phillips and Sumners 1998; Veerasingham and Raizada 2003; Guyenet 2006; Zucker 2006)
Exogenous AngII Stability in Neuron Culture Media cardiovascular function, including the subfornical organ (SFO), paraventricular nucleus (PVN), and rostral ventrolateral medulla (RVLM), AngII increases neuronal firing leading to the deleterious sympathoexcitation commonly associated with neurocardiovascular diseases (Bains et al 1992; Ferguson and Bains 1997; Gao et al 2005; Feng et al 2008)
AngII can activate its type 2 receptor (AT2R) on central neurons, which often results in opposing responses compared to AT1R activation, reflecting the different physiological and pathophysiological roles of AngII mediated via these receptors (Sumners et al 1994)
Summary
Dysregulation of angiotensin II (AngII)-dependent neuronal signaling in the central nervous system is involved in the pathogenesis of cardiovascular diseases, such as hypertension and chronic heart failure (Phillips and Sumners 1998; Veerasingham and Raizada 2003; Guyenet 2006; Zucker 2006). Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society AngII stimulation of its type I receptor (AT1R) in these cardiovascular control brain regions and others contributes to vasopressin secretion, thirst and salt appetite, and baroreflex modulation (Matsukawa et al 1991; Reid 1992; Johnson and Thunhorst 1997). AngII can activate its type 2 receptor (AT2R) on central neurons, which often results in opposing responses compared to AT1R activation, reflecting the different physiological and pathophysiological roles of AngII mediated via these receptors (Sumners et al 1994)
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