Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy.
Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.
Department of Chemistry, University of Washington, Seattle 98195-1700, USA.
P S
PS
Hodder
C
C
Beeson
J
J
Ruzicka
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D007202:Indicators and Reagents; D007700:Kinetics; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D051381:Rats; D011976:Receptors, Muscarinic / Q000737:chemistry*; D013053:Spectrophotometry
D007202:Indicators and Reagents; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic