Assays for intracellular cyclic adenosine monophosphate (cAMP) and lysosomal acidification

Abstract

Cyclic adenosine monophosphate (3′,5′-cAMP) is a multifunctional second messenger which controls extremely diverse and physiologically important biochemical pathways. Among its myriad roles, 3′,5′-cAMP functions as an intracellular regulator of lysosomal pH, which is essential for the activity of acidic lysosomal enzymes. Defects in lysosomal acidification are attributed to many diseases like macular degeneration, Parkinson’s, Alzheimer’s, and cystic fibrosis. Strategic re-acidification of defective lysosomes by pharmacological increase of intracellular cAMP offers exciting therapeutic potential in these diseases. Modular assays for accurate assessment of intracellular cAMP and lysosomal pH are a critical component of this research. We describe label-free targeted metabolomics for quantitating intracellular cAMP and integrated assays for measuring lysosomal pH. These hybrid assays offer fast, unbiased information on intracellular cAMP concentrations and lysosomal pH that can be applied to many cell types and putative drug screening strategies.