Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry

Human secretin receptor is a G protein-coupled receptor that is functionall y linked to the cAMP second messenger system by stimulation of adenylate cy clase. To functionally characterize the receptor and evaluate its signal tr ansduction pathway, the full-length human secretin receptor cDNA was subclo ned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidifica tion rate was measured by the Cytosensor microphysiometer. Human secretin a nd biotinylated human secretin were equipotent in both assays in a dose-dep endent manner. The EC50 values of stimulating the intracellular cAMP accumu lation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmen ted by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicat ing that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the prote in kinase A inhibitor H-89, suggesting that protein kinase A plays an essen tial role in the intracellular signaling of the receptor. Upon repeated sti mulation by the ligand, the peak acidification responses did not change sig nificantly at both physiological (0.03 nM and 3 nM) and pharmacological (0. 3 mu M) concentrations of human secretin, suggesting that the human secreti n receptor did not exhibit robust homologous desensitization. J. Cell. Bioc hem. 72:51:7-527, 1999. (C) 1999 Wiley-Liss, Inc.