Abstract 152: Development of a Chronic Angiotensin II (AngII)-stimulated Neuronal Cell Culture Model

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Dysregulation of AngII-mediated intra-neuronal signaling contributes to the pathogenesis of hypertension. Previous studies using AngII-sensitive neuronal cell culture models suggest that AngII induces chronic changes in the expression of various cell signaling proteins, including its own receptors. However, many of these studies stimulated cultured neurons with a single administration of exogenous AngII and harvested neurons 3-24 hours later to investigate changes in mRNA and protein levels. One limitation of these “chronic” AngII stimulation studies is the lack of evidence indicating the stability of AngII in neuronal cell culture media. Here, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Further, we hypothesized that AngII receptors are differentially expressed depending on the chronicity of stimulation. AngII (100nM) was added to catecholaminergic (CATH.a) neurons in culture, media was collected 15 min - 24 hr later, and AngII levels were determined by liquid chromatography-tandem mass spectrometry. AngII was rapidly metabolized with media levels of AngII returning to near baseline within 3 hr of administration (baseline: 2.2 nM AngII; 15 min: 50.6 nM; 30 min: 42.9 nM; 1hr: 28.8 nM ; 3hr: 4.0 nM, n = 3-4). To begin investigating the relevance of this observation, we measured mRNA levels of AngII type 1 (AT1R) and type 2 (AT2R) receptors using real time quantitative RT-PCR in CATH.a neurons 24 hr after a single administration of AngII (100nM) or repeated administration every 3 hr. While a single administration of AngII did not alter AT1R mRNA levels, replenishing media every 3 hr with fresh AngII did induce a 20% decrease. In addition, fresh AngII every 3 hr resulted in a 41% decrease in AT2R mRNA levels; whereas, a single dose of AngII increased AT2R mRNA by 19%. These data indicate that AngII is quickly metabolized in neuronal cell culture media, and that supplementing fresh AngII every 3 hr to maintain elevated levels chronically yields different changes in AngII receptor mRNA levels as compared to a single administration. In conclusion, we suggest that to accurately model hypertension, where AngII is chronically elevated, the metabolism of AngII in a given cell culture model should first be determined.

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