A calcium fluorescence assay for quantification of cholinergic receptor activity of clinical drugs in serum -...

A new approach is described for quantifying cholinergic receptor activation status human blood samples, based on M1 receptor-driven mobilization of intracellular calcium stores. The assay identifies anticholinergic as well as agonist cholinergic receptor activity. As a cell-based procedure, the assay shares the high efficiency of recently developed M1 receptor binding protocols, but differs from the latter in relying on fluorescence rather than radioactivity measurements. The assay targets a true functional effect insofar as it reflects a time-dependent process of net changes in activation of cholinergic receptors. Results from experiments with M1-expressing CHO cells exposed to a fluorogenic dye and the standard cholinergic agonist carbachol revealed the assay's ability to isolate pure agonist effects of clinical compounds as well as the net effects of serum containing agonist and antagonist factors. The new protocol thus provides two additional quantitative indices of cholinergic receptor activity in human serum, namely pure agonistic effects and net agonist/antagonist effects. As such, it could constitute a very useful addition to efforts to quantify global cholinergic status in human serum in various clinical conditions. By relying on fluorescence measures it should also prove much more accessible than radioactivity-based protocols.