G protein-coupled receptors (GPCRs) signal in response to various external stimuli including stress. GPCR signaling has been shown to play a critical role in the adaptation of cell response to limited oxygen supply. Hypoxia has been implicated in cardiovascular diseases, human pulmonary arterial responses, and persistent pulmonary hypertension in newborns. One of the key GPCRs implicated in hypoxia is the prostanoid receptor, thromboxane A2 receptor (TP). Hypoxia can affect TP localization, stability, and activity both in vivo and in vitro. To elucidate hypoxia-mediated GPCR signaling in vitro, we lay out a general strategy to perform hypoxic experiments using both primary pulmonary artery smooth muscle cells and TP expressed in HEK293T cells. We describe assay for measuring moderate tissue hypoxia using static cell cultures, monitoring pericellular media oxygen content, and signaling of TP.
Keywords: G protein-coupled receptor; Hypoxia; Prostanoid receptor; Stress response; Thromboxane A2 receptor.
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