Angiotensin II (AngII) is rapidly metabolized in neuronal cell culture media as determined by liquid chromatography‐tandem mass spectrometry (LC MS/MS)

Abstract

Previous reports using AngII‐sensitive neuronal cell culture models suggest that AngII induces chronic changes in various cell signaling proteins (e.g. kinases & transcription factors). Many of these studies stimulated cultured neurons with a single administration of exogenous AngII and harvested neurons 1–3 days later to investigate changes in protein levels. One limitation of such “chronic” AngII stimulation studies is the lack of evidence indicating the length of time exogenous AngII remains in neuronal cell culture media. Herein, using LC MS/MS, we tested the hypothesis that exogenous AngII is quickly metabolized to other angiotensin peptides in neuronal cell culture media. AngII (104.62pg/μl) was added to cultured CATH.a neurons and media was collected 15 min – 24 hr later for LC MS/MS analysis. Media levels of AngII rapidly and significantly decreased over time (52.8pg/μl AngII at 15 min; 4.2pg/ul at 3 hr; 0.37pg/μl at 24 hr, p<0.05, n = 3–4). At early time points (15 min – 3 hr), levels of AngIII and Ang1–7, two peptides generated from AngII, increased with a subsequent decrease at later times. Levels of AngI and AngIV did not significantly change. These studies provide new insight into the half‐life and metabolism of AngII in neuronal cell culture media and suggest previous studies using a single administration of AngII to study “chronic” AngII intra‐neuronal signaling pathways be reevaluated. NIH R01HL103942