Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay

Background

Cyclic 3’,5’-adenosine monophosphate (cAMP) is a ubiquitously expressed second messenger, synthesised by adenylate cyclase enzymes (ACs), that acts as a master regulator of a broad spectrum of intracellular signalling pathways (Hayes and Brunton, 1982; Beavo and Brunton, 2002). Homeostatic regulation of cAMP is fine-tuned by phosphodiesterase enzymes (PDEs), rapidly degrading cAMP into 5’AMP (Baillie, 2009). PDEs consist of 11 families (PDE1-11) spanning over 100 isoenzymes (e.g., PDE4A1-PDE4A5), all with the ability to rapidly hydrolyse cyclic nucleotides (Conti and Beavo, 2007). Of these, PDE1-4, 7, 8, 10 and 11 possess the ability to catalyse the hydrolysis of cAMP. Compartmentalisation of specific PDE isoforms within discrete signalosomes is crucial to the spatio-temporal control of cAMP nanodomains, tightly regulating cAMP-effector protein activity (Blair and Baillie, 2019). In light of this, it is no surprise that aberrant PDE activity plays a central role in disease pathogenesis, with many PDEs being identified as a driving force in cardiovascular disease (Bobin et al., 2016), respiratory disease (Zuo et al., 2019), neurological disease (Knott et al., 2017) and cancers (Peng et al., 2018). Thus, research and development into novel and effective therapies targeting PDE activity remains a highly competitive area in drug discovery (Maurice et al., 2014; Baillie et al., 2019). This demonstrates a clear need for the development of assays with the ability to measure PDE activity, not only as a tool to investigate PDE activity/function in cellular signaling, but as a means of assessing and developing therapies targeting PDE activity.

Marchmont and Houslay (1980) previously described a method of measuring PDE activity, modified from a two-step procedure developed by Thompson and Appleman (1971). This radioassay directly quantifies the formation of radioactively tagged 8-[³H] adenosine, formed as a result of cAMP hydrolysis by PDE within a given sample, allowing for the rate of cAMP hydrolysis to be determined. This highly sensitive assay has proven to be an invaluable tool in the understanding of unique PDE functionality and in assessing the effectiveness of novel PDE therapies (example studies utilising this method: Schafer et al., 2010; Moretto et al., 2015; Omar et al., 2019; Houslay et al., 2019). Here we present a straight-forward and effective procedure for indirectly measuring the activity of cAMP hydrolysing PDEs (Figure 1).


Figure 1. Simple schematic representing Phosphodiesterase Assay procedure