Assessment of Extracellular ATP Concentrations
Most cells release ATP to the extracellular milieu. Extracellular ATP plays important signaling roles by activating a score of broadly distributed cell surface purinergic receptors (purinoceptors). Biological responses regulated by purinergic receptors include neurotransmission, smooth muscle relaxation and contraction, epithelial cell ion transport, inflammation, platelet activation, immune responses, cardiac function, endocrine and exocrine secretion, glucose transport, and cell proliferation. ATP concentrations at the cell surface, and consequently the magnitude of purinergic receptor stimulation, reflect a well-controlled balance between rates of ATP release and extracellular metabolism. Given the broad spectrum of responses triggered by extracellular ATP, there is a growing interest in accurately assessing the concentrations of this nucleotide at the cell surface. In this chapter, we discuss the use of the luciferin/luciferase-based reaction to measure extracellular ATP concentrations with high sensitivity. Protocols are adapted to assess ATP levels either in sampled extracellular fluids or in situ at the cell surface. Although our focus is on studies of ATP release from epithelial cells, protocols described here are applicable to practically all cell types.
Key wordsATP release extracellular ATP ecto-ATPase luciferase protein A-luciferase luciferin
- 6 × His
arbitrary light unit
bovine serum albumin
Dulbecco’s modified eagle’s medium
fetal bovine serum
Hank’s balanced salt solution
4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid
minimum essential medium
Staphylococcus protein A-fused luciferase.